Host cells transformed with the E. coli glucoronide permease gene

ABSTRACT

The present invention relates to the β-glucuronidase (GUS) gene fusion system, and to the cloning and characterization of the β-glucuronidase and glucuronide permease genes of Escherichia coli. It is based on the surprising discovery that gene fusions comprising the β-glucuronidase gene may be effectively expressed in a wide variety of organisms to produce active β-glucuronidase enzyme. Because of the abundance and availability of useful substrates for β-glucuronidase enzyme, GUS gene fusions may serve as a superior reporter gene system as well as an effective means of altering cellular phenotype. In conjunction with recombinant glucuronide permease, which may be used to render host cells permeable to β-glucuronidase substrates, the GUS gene fusion system offers almost unlimited applications in the fields of plant and animal genetic engineering.

This application is a division of application Ser. No. 07/447,976 filedDec. 8, 1989, now U.S. Pat. No. 5,268,463 granted Dec. 7, 1993; which isa continuation-in-part of application Ser. No. 07/264,586 filed Oct. 31,1988 (abandoned); which is a continuation-in-part of application Ser.No. 07/119,102 filed Nov. 10, 1987 (abandoned); which claims priorityunder 35 U.S.C. § 119 to British application 8725402 filed Oct. 29, 1987and British application Ser. No. 8626862 filed Nov. 11, 1986.

TABLE OF CONTENTS

1. Introduction

2. Background of the Invention

2.1. Beta-Glucuronides 2.2. Beta-Glucuronidase 2.3. The Utility of GeneFusion Systems

2.3.1. A Review of Existing Gene Fusion Systems

SUMMARY OF THE INVENTION 3.1. Abbreviations and Definitions 4.DESCRIPTION OF THE FIGURES 5. DETAILED DESCRIPTION OF THE INVENTION 5.1.Cloning and Characterization of Beta-Glucuronidase Genes 5.2. Cloningand Characterization of Glucuronide Permease Genes 5.3. Promoters ThatMay Be Useful in the Beta-Glucuronidase Gene Fusion System

5.3.1. Introduction of Beta-Glucuronide Permease and/or Permease into aHost Cell or Organism

5.4. Utility of Beta-Glucuronidase Gene Fusions as Reporter Genes 5.5,Utility of Beta-Glucuronidase Gene Fusions in the Manipulation ofCellular Phenotype 5.6. Utility of Glucuronide Permease 5.7. AdditionalUses of the GUS System 5.8. Useful Substrates for the Beta-GlucuronidaseGene Fusion System 5.9 Methods of Analysis of GUS Expression

5.9.1. Lysis and Extraction

5.9.2. Composition of Extraction Buffers

5.9.3. Protease Action on Gus

5.9.4. Storage of Extracts

5.9.5. Treatment of Extracts to Reduce Endogenous Fluorescence orAbsorption

5.9.6. β-Glucuronidase Assays

5.9.6.1. Fluorogenic Assays

5.9.6.2. Spectrophotometric Assay

5.9.7. Histochemical Assays of GUS

6. EXAMPLE: CLONING OF THE ESCHERICHIA COLI GENE FOR BETA-GLUCURONIDASE6.1 Materials And Methods

6.1.1. DNA Manipulation

6.1.2. Protein Sequencing and Amino Acid Analysis

6.1.3. Protein Analysis

6.1.4. Beta-Glucuronidase Assays

6.1.5. Purification of Beta-Glucuronidase

6.2. Results

6.2.1. Subcloning and Sequencing of the uidA Gene

6.2.2. Manipulation of the uidA Gene for Vector Construction

6.2.3. Purification and Properties of Beta-Glucuronidase

6.3. Discussion

6.3.1. Molecular Analysis of the uid Locus

6.3.2. The uidA Gene as Gene Fusion Marker

7. EXAMPLE: EXPRESSION OF BETA-GLUCURONIDASE GENE FUSIONS CAENORABDITISELEGANS 7.1. Materials

7.1.1. DNA Constructs

7.1.2. Transformation with Plasmid DNA

7.1.3. Fluorometric Assays

7.2. Results and Discussion 8. EXAMPLE: EXPRESSION OF BETA-GLUCURONIDASEGENE IN HIGHER PLANTS 8.1. Materials and Methods

8.1.1. Nucleic Acid Manipulation

8.1.2. Plant Transformation and Regeneration

8.1.3. Southern Blot Analysis

8.1.4. Substrates

8.1.5. Lysis Conditions

8.1.6. Spectrophotometric Assay

8.1.7. Fluorometric Assay

8.1.8. In Situ Localization of GUS Activity in SDS Polyacrylamide Gels

8.1.9. Histochemical Assay

8.1.10. Purification of Beta-Glucuronidase

8.2. Results

8.2.1. Higher Plants Contain no Detectable Beta-Glucuronidase Activity

8.2.2. Construction of Plasmids for Transformation of Plants with GUSFusions

8.2.3. Chimeric GUS Genes are Expressed in Transformed Plants

8.2.4. Visualization of GUS Activity on SDS-Polyacrylamide Gels

8.2.5. GUS Activity in Plants can be Visualized using HistochemicalMethods

8.3. Discussion EXAMPLE: CLONING EXPRESSION OF THE ESCHERICHIA COLIGLUCURONIDE PERMEASE GENE 9.1. Materials and Methods

9.1.1. Plasmids and DNA

9.2. Results and Discussion

9.2.1. Locating the Glucuronide Permease Coding Region

9.2.2. Analysis of Amino Acid Sequence and the Glucuronide PermeaseProtein

9.2.3. Molecular Genetic Demonstration of Glucuronidase Permease

10. EXAMPLE: TRANSGENIC PPLANTS EXPRESSING A BETA-GLURONIDASE GENEFUSION AND ALTERNATION OF GROWTH PATTERNS BY AUXIN-GLUCURONIDASE 10.1.Materials and Methods 10.2. Results and Discussion 11. EXAMPLE: THE USEOF GUS FUSIONS IN TRANSGENIC PLANTS: REGULATION OF CHIMERIC PATATINGENES IN TRANSGENIC POTATO PLANTS 11.1. Materials and Methods 11.2.Results and Discussion

11.2.1. In Vitro Induction Experiments

11.2.2. Patatin-GUS Expression in Planta

11.2.3. Design of the Field Trial

11.2.3.1. GUS I

11.2.3.2. GUS II

11.2.3.3. GUS III

11.2.3.4. Containment Consideration

11.2.3.5. Planting, Growth and Harvest Procedure

11.2.3.6. Sampling and Assay of GUS Activity

11.2.3.7. Results from the Field Analysis

12. EXAMPLE: THE ENZYMATIC ACTIVITY ASSAY OF THE β-GLUCURONIDES INDUCEDBY VARIOUS GLUCURONIDES 13. DEPOSIT OF MICROORGANISMS

1. Introduction

The present invention relates to the β-glucuronidase (GUS) gene fusionsystem, and to the cloning and characterization of the β-glucuronidaseand glucuronide permease genes of Escherichia coli. It is based on thesurprising discovery that gene fusions comprising the β-glucuronidasegene may be effectively expressed in a wide variety of organisms toproduce active β-glucuronidase enzyme. Because of the abundance andavailability of useful substrates for β-glucuronidase enzyme, GUS genefusions may serve as a superior reporter gene system as well as aneffective means of altering cellular phenotype. In conjunction withrecombinant glucuronide permease, which may be used to render host cellspermeable to β-glucuronidase substrates, the GUS gene fusion systemoffers almost unlimitted applications in the fields of plant, microbialand animal genetic engineering.

2. Background of the Invention

In mammals, glucuronidation is a principle means of detoxifying orinactivating compounds which utilizes the UDP glucuronyl transferasesystem. In humans, a number of hormones, including cortisol andaldosterone testosterone and androsteindione, certain antibiotics suchas chloramphenicol, toxins such as dinitrophenol, and bilirubin areamong the compounds which are conjugated to form glucuronides by theglucuronyl transferase system and then excreted in urine or into thelower intestine in bile. The bacterium Escherichia coli has evolved tosurvive in the mammalian intestine, and can utilize the excretedβ-glucuronides as its sole carbon source. To do so, E. coli has evolvedmechanisms for the uptake and degradation of a wide variety ofglucuronides, processes which are tightly linked genetically.

2.1. Beta-Glucuronides

Most aromatic and aliphatic glucuronides are remarkably stable relativeto other types of glycoside conjugates. It is speculated that this isdue to the inductive effect of the carbonyl group at C-6 on thehemiacetal linkage at C-1. Many β-glucuronides can be prepared free ofother contaminating glycosides by vigorous acid hydrolysis, whichcleaves glucosides, galactosides and other glycosides, but leaves mostglucuronides intact. For example, complex carbohydrate polymers such asgum arabic can be reduced to a collection of monosaccharide components,and the single β-glucuronyl disaccharide aldobiuronic acid, simply byboiling gum arabic in sulfuric acid overnight. Colorigenic andfluorogenic glucorogenic substrates such as p-nitrophenylβ-D-glucuronide and 4-methylumbelliferyl β-D-glucuronide are much morestable in aqueous solution than the corresponding β-D-galactosides orβ-D-glucosides.

β-Glucuronides in polysaccharide form are common in nature, mostabundantly in vertebrates, where in polymeric form with other sugarssuch as N-acetylglucosamine they are major constituents of connectiveand lubricative tissues (e.g. chondroitin sulfate of cartilage, andhyaluronic acid, the principle constituent of synovial fluid and mucus).β-glucuronides are relatively uncommon in plants. However, some plantgums and mucilages produced by wounded trees, notably gum arabic fromAcacia senegal, do contain significant fractions of β-glucuronides inpolymeric form, although rarely if ever as terminal residues.Glucuronides and galacturonides found in plant cell wall components(such as pectin) are generally in the alpha configuration, and arefrequently substituted as the 4-O-methyl ether; hence these are notsubstrates for β-glucuronidase.

As simple glycosides, β-glucuronides are extremely important as theprinciple form in which xenobiotics and endogenous phenols and aliphaticalcohols are excreted in the urine and bile of vertebrates (reviewed byDutton, G. J., 1966, ed., Glucuronic Acid, Free and Combined, N.Y.;Dutton, G. J., 1980, Glucuronidation of Drugs and Other Compounds,Florida). Detoxification of xenobiotics by glucuronidation is the mostimportant mechanism for elimination of inappropriate compounds from themetabolism of vertebrates. Glucuronidation occurs in many tissues invertebrates, most notably in the liver. The reaction is carried out by aset of membrane-bound enzymes catalyzing the transfer of a glucuronateresidue from uridine diphosphate 1-alpha-D-glucuronate to the aglycone(xenobiotic). There are several isozymes of UDP-glucuronyl transferasethat have been characterized (for a thorough review, see Dutton, 1980,supra). These enzymes are frequently part of a suite of detoxifyingenzymes, including hydroxylases and mixed-function oxidases that work inconcert to metabolize lipophilic, relatively insoluble compounds intothe highly water-soluble glucuronide conjugates (as well as sulfates andother derivatives). These conjugates are then excreted into the bile(for the larger glucuronide conjugates) or the urine. Several thousandβ-glucuronides have been characterized in urine and bile asdetoxification products, many following administration of the freeaglycone or a related compound (a compendium of many glucuronides can befound in Dutton, 1966, supra). In addition, many endogenous steroidhormones and bioactive substances, or bio-degradation products such asbilirubin, are conjugated and excreted as β-glucuronide conjugates. Thisextremely important and voluminous pathway and the fact that, amongenteric bacteria, the β-glucuronidase gene appears almost exclusivelylimited to E. coli may account in part for the success of E. coli as aprinciple and ubiquitous colonizer of the vertebrate intestine andurinary tract.

Interestingly, β-glucuronidase activity is reliably reported almostexclusively from those organisms that have, or are associated withorganisms that have glucuoridation as a detoxification pathway. Thusvertebrates, which all use glucuronidation as the principle conjugationmechanism, together with some of their endogenous microbe populations(usually E. coli) have GUS activity. By contrast, insects and plantsconjugate xenobiotics with glucose, rather than glucuronic acid, astheir detoxification and derivatization mechanism, and β-glucuronidaseis rarely if ever reported in these organisms or their attendantmicrobial populations.

2.2. Beta-Glucuronidase

Beta (β)-glucuronidase (GUS) catalyzes the hydrolysis of a very widevariety of β-glucuronides, and, with much lower efficiency, hydrolyzessome β-galacturonides; the reaction and a small selection of theavailable substrates for routine assay of the enzyme are diagrammed inFIG. 13. Almost any aglycone conjugated in a hemiacetal linkage to theCl hydroxyl of a free D-glucuronic acid in the β configuration serves asa GUS substrate. Glucuronides are generally very water soluble, due tothe ionizable carboxylic acid group at the 6-carbon position in theglycone.

E. coli β-glucuronidase (GUS) has a monomer molecular weight of about68,200 daltons, although under certain conditions of SDS-polyacrylamidegel electrophoresis it migrates a bit slower than would be predicted(around 72-74 kDa). The behaviour of the native enzyme on gel filtrationcolumns indicates that the active form is probably a tetramer. It is notprocessed at the amino terminus in E. coli, and is found exclusively inthe cytoplasm. GUS is an exo-hydrolase; it will not cleave glucuronidesin internal positions within polymers. GUS is specific forβ-D-glucuronides, with some tolerance for β-galacturonides. It isinactive against β-glucosides, β-galactosides, β-mannosides, orglycosides in the alpha configuration.

β-Glucuronidase is very stable, and will tolerate many detergents andwidely varying ionic conditions. It is most active in the presence ofthiol reducing agents such as β-mercaptoethanol or dithiothreitol (DTT).GUS has no cofactors, nor any ionic requirements. GUS is inhibited bysome divalent metal ions: 70% inhibition by Mn²⁺ and Ca²⁺ at 10 mM, andcompletely by Cu²⁺ and Zn²⁺ at comparable concentrations (Stoeber, 1961,Etudes des propietes et de la biosynthesase de la glucuronidase et de laglucuronide-permease chez E. coli. These de Docteur es Sciences, Paris).β-Glucuronidase can be assayed at any physiological pH, with an optimumbetween 5.0 and 7.8. The enzyme is about 50% as active at pH 4.3 and atpH 8.4. GUS from E. coli K12 is reasonably resistant to thermalinactivation with a half-life at 55° C. of about two hours and at 60°C., about 15 minutes. The specific inhibitor, glucaric acid 1,4 lactone(saccharic acid lactone, saccharolactone) is a very useful reversiblecompetitor inhibitor of GUS.

β-Glucuronidase activity is extremely common in almost all tissues ofall vertebrates and many molluscs (Levvy, G. A. and Conchie, J., 1966,β-Glucuronidase and the hydrolysis of glucuronides, in Glucuronic Acid,Free and Combined, N.Y. p. 301). The enzyme has been purified from manymammalian sources (e.g. Tomino et al., 1975, J. Biol. Chem. 250:8503)and shows a homotetrameric structure, with a subunit molecular weight ofapproximately 70 kDa. The enzyme from these sources is synthesized witha signal sequence at the amino terminus, and is then transported to andglycosylated within the endoplasmic reticulum and ultimately localizedwithin vacuoles intracellularly. Unlike the bacterial enzyme, mammalianand molluscan GUS can cleave thioglucuronides. In general, however, theE. coli GUS is much more active than the mammalian enzyme against mostbiosynthetically derived β-glucuronides (Tomasic, J. and Keglevic, D.,1973, Biochem. J. 133:789-795; and Levvy and Conchie, supra). Thegenetics of GUS in mammals have been extensively characterized (reviewedin Paigen, K., 1979, Ann. Rev. Genet. 13:417-466).

GUS activity is largely if not completely absent from higher plants(Jefferson et al., 1987, EMBO J. 6:3901-3907) mosses, algae and ferns.There are a few reports of endogenous activity in plants but they-rarelyinclude quantitative tests with more than one substrate, to ensure thatthe activity is a true β-glucuronidase, not an activity specific for theaglycone of the test substrate (e.g., Schultz, M. and Weissenbock, G.,1987, Phytochemistry 26:933-938), nor do they often make use of specificinhibitors of GUS such as saccharolactone (see below). Such reportsshould also be interpreted cautiously because only rarely do plantsexist without numerous exo- and endophytic organisms, many not yetclassified, which could be contributing GUS activity. Specificglucuronidase that recognize endogenous substrates such as glycyrrhizinconjugates have been described, but are not capable of cleaving GUSassay substrates.

The free-living soil nematode, Caenorhabditis elegans, has an endogenousβ-glucuronidase activity which occurs at low levels in the intestine ofthe worm. Enzyme activities in the other nematodes have apparently notbeen investigated.

Very few insects have been investigated for intrinsic GUS activity.Studies on Drosophila melanogaster embryos, pupae and larvae showed nodetectable activity under conditions that gave very high levels ofβ-galactosidase (Jefferson, 1985, (published 1986) DNA Transformation ofCaenorhabditis elegans: Development and Application of a New Gene FusionSystem. PhD. Dissertation, University of Colorado, Boulder). Extractsfrom white flies and black flies from glasshouse populations alsorevealed very little if any GUS activity. Locust crop fluid liquor is asource of GUS but it is not clear whether this is an intrinsic activity,or due to microorganisms in the crop fluid.

GUS activity has not yet been found in any fungi, includingSaccharomyces, (Jefferson, 1985, DNA Transformation of Caenorhabditiselegans: Development and Application of a New Gene Fusion System. PhD.Dissertation, University of Colorado, Boulder; and Schmitz et al., 1989,Gene, in press) Schizosaccharomyces, Aspergillus, Neurospora,Cladosporium, Leptosphaeria and other Ascomycetes such as barley powderymildew or Oomycetes such as Bremia lactuca. There is also no detectableactivity in the slime mould, Dictyostelium discoidium (Datta et al.,1986, Molec. Cell. Biol. 6:811-820; and Jefferson, 1985, DNATransformation of Caenorhabditis elegans: Development and Application ofa New Gene Fusion System. PhD. Dissertation, University of Colorado,Boulder).

GUS is not present in most bacterial genera examined, includingBacillus, Klebsiella, Proteus, Erwinia, Rhizobium, Bradyrhizobium,Agrobacterium, Pseudomonas, Xanthomonas, Anabaena and Actinomycetesalthough it must be remembered that induction of genes for GUS isrequired before activity can be found even in definitively GUS⁺bacteria. The intestinal commensal Enterobacteriaceal species E. coli isone of the only species of bacteria that has been found reliably to havea β-glucuronidase activity; in fact, the presence of β-glucuronidase isa widely accepted diagnostic test for E. coli in natural populations ofbacteria isolated from sources such as urine, feces, contaminated wateror food (e.g. Godsey, et al., 1981, J. Clin. Microbiol. 13:483-490;Feng, P. C. S. and Hartman, P. A., 1982, Appl. Environ. Microbiol.43:1320-1329; Trepeta, R. W. and Edberg, S.C., 1984, J. Clin. Microbiol.19:172-174; and Moberg, L. F., 1985, Appl. Environ. Microbiol.50:1383-1387). The GUS activity of E. coli populations in the intestineplays a very significant role in the physiology of most vertebrates,being partially or wholly responsible for enterohepatic recirculation ofconjugated drugs, hormones and xenobiotics. Recent work indicates thatthere is at least one other genus of bacterium (Alcaligenes sp.) foundin soil and water samples, and in urine and feces, that appears toproduce GUS upon induction. Regions of the E. coli chromosome,containing portions of the GUS operon have been subcloned into E. coliplasmids (Blanco et al., 1982, J. Bacteriol. 149:587-594); however,prior to the present invention, the gene encoding GUS had not beenisolated and characterized, nor had the coding system been expressed ina heterologous system.

In addition to β-glucuronidase enzyme, the GUS operon of E. coli alsoencodes glucuronide permease, first described biochemically by F.Stoeber (1961, These de Docteur des. Sciences, Paris). Glucuronidepermease provides a mechanism for transport of β-glucuronidase throughthe cell membrane, and permits the entrance of a surprisingly widevariety of substrates, ranging from simple aliphatic compounds to large,complex heterocyclic conjugates (FIG. 13), into the cytoplasm.β-galactoside permease, in contrast, will only admit very simplemolecules but not compounds of any appreciable complexity (e.g. complexphenolic compounds or heterocyclic compounds such as x-gal.

The combination of GUS and glucuronide permease enables E. coli toutilize a vast repertoire of glucuronides as sources of energy. Numerouscompounds (including hormones, cholesterol, and antibiotics) areconjugated to glucuronic acid in the human liver, and thereby nourishthe bacteria that constitute the intestinal flora. In turn, bymetabolizing these compounds, E. coli significantly impacts on thebioavailability of a multitude of biologically relevant molecules.

2.3. The Utility of Gene Fusion Systems

"Reporter" genes are used in molecular biology as indicators of geneactivity. A reporter gene will typically encode an enzyme activity thatis lacking in the host cell or organism which is to be transformed. Thisallows the measurement or detection of the enzyme activity which may beused as an indicator or "reporter" of the presence of expression of thenewly introduced gene.

A reporter gene may be put under the influence of a "controller"sequence, such as a promoter element. Successful expression of reportergene product serves as an indicator of controller element activity.Additionally, a reporter gene may be used as a DNA transformationmarker. Cells may be transformed with DNA comprising a gene of interestwhich encodes a product that is difficult or impossible to detect aswell as DNA comprising the reporter gene under the control of a suitabletranscriptional promoter; expression of reporter gene activity in a cellis suggestive of successful transformation with the gene of interest,the presence of which may be corroborated by standard moleculartechniques. Measurement of reporter enzyme activity is frequently usedto infer characteristics of the transcription of a gene encoding thereporter enzyme. These inferences depend on several assumptions thatshould be examined, and, when possible, controlled experimentally.Firstly, we should be aware that the ultimate regulated level of a geneproduct is determined by a large number of factors, only one of which isthe initiation of transcription. While transcription does appear to bethe principle site of regulation of gene action, there are numerousother components in the regulatory pathway that must be considered, andin some situations they will prove to be more important thantranscriptional control. For instance, it is clear that DNAmodifications such as methylation, chromatin configuration and possiblythree dimensional structure and location of the gene can influence itsexpression. It is also obvious that control of precursor RNA processingand transport--including the correct excision of introns,polyadenylation of the transcript, extranuclear transport to a site oftranslation, and degradation of the mRNA or its precursors can haveprofound effects on the eventual levels of a protein product. Thefrequency of translational initiation, the rate of extension, theprocessing, modification and/or targeting or the primary protein productas well as its degradation and turnover will also inevitably affectfinal product levels.

The use of precise gene fusions can simplify analysis of this complexprocess. For example, it is possible to delineate the contribution oftranscriptional control of gene expression by eliminating all thespecific signals for post-transcriptional controls and replacing themwith sequences from a readily assayed responder gene. Further carefulgene fusion constructions can then be performed to assay the effects ofinclusion of additional controller sequences, for instance the"untranslated leader sequences" or the sequences surrounding the site oftranslational initiation. Gene fusions need not be confined to promoteranalysis, since factors affecting mRNA processing and stability, (suchas polyadenylation signals or introns), or translational efficiency(such as the context of the initiator codon or mRNA secondarystructure), will inevitably affect reporter enzyme levels. With theappropriate controls, many of these regulatory steps can also beanalyzed with gene fusion technology. It is important to be aware of thepotential contributions of these "downstream" points in the regulatorypathway of gene expression so they can be considered in the design andinterpretation of gene fusion experiments.

In addition, many genes in plants and other higher organisms exist inmulti-gene families whose products are very similar but can be regulateddifferentially during development; in fact many times members ofmulti-gene families are apparently inactive. By using gene fusions toindividual members of such families and introducing these fusions intothe genome, one can study the expression of individual genes separateand distinct from the background of the other members of the genefamily.

Analysis of mutationally altered genes in plants accessible totransformation techniques is greatly facilitated by the use of sensitiveand versatile reporter enzymes. Many of the regulatory parametersresponsible for spatial and temporal restriction of gene activityrequire specialized analytic methods. Moreover, the logistics ofanalyzing gene function in large numbers of transgenic plants can beoverwhelming, unless routine, high resolution techniques are available.Although many of the plant genes that have been characterized to dateproduce abundant products that are measurable by existing means, manymore will certainly be described whose products are of moderate or lowabundance; these will doubtlessly prove important and interesting tostudy, requiring increasingly sensitive methods. By using a reportergene that encodes an enzyme activity not found in the organism beingstudied, the sensitivity with which chimeric gene activity can bemeasured is limited only by the properties of the reporter enzyme andthe quality of the available assays for the enzyme.

2.3.1. A Review of Existing Gene Fusion Systems

An ideal gene fusion system should provide a reporter enzyme that isstable, tolerates amino terminal fusions, has numerous, simple andversatile assays, and that has no intrinsic background activity in theorganism being studied. Furthermore, the enzyme should not interferewith normal physiological functioning of the organism, nor affect thebiochemistry adversely. The assays should be sensitive enough to measuregene expression of moderate to low abundance in single cells, and shouldallow spatial discrimination of enzyme activity within the complexcellular patterns of tissues and organs. In addition, the assays shouldbe quantitative, inexpensive and uncomplicated. The enzyme should beactive under widely varying conditions of pH, ionic environment andtemperature, and should be tolerant of general laboratory manipulations.There should also be the possibility of using the system as a trueresponder, providing both reporting and effecting functions, therebyallowing genetic selections to be applied. There should be methodsavailable to use the system in live organisms quantitatively. Progressin agricultural molecular biology, and especially the use of genefusions in transgenic plants, fungi and bacteria of agricultural andindustrial importance will be greatly enhanced by the availability ofsuitable responder genes encoding enzymes with this set of properties.

At least seven reporter genes have been used in studies of geneexpression in higher plants. These include the E. coli β-galactosidase(lacZ, LAC), chloramphenicol acetyl transferase (CAT), neomycinphosphotransferase (APH3'II, NPTII), nopaline synthetase (NOS), octopinesynthase (OCS), firefly luciferase (luc) and bacterial luciferase (luxAand luxB). Each of these systems has properties that make them less thanoptimal for gene fusion analysis.

The lacZ gene from E. coli is part of an operon of three genes--the lacoperon--and encodes a stable β-galactosidase with a wide substratespecificity. This gene was the first used in gene fusion experiments inthe construction of the trp-lac fusion in the E. coli chromosome, wellbefore the advent of recombinant DNA manipulations in vitro (Beckwith etal., 1967, Transposition of the lac region of E. coli, Cold SpringHarbor Symp. Quant. Biol. 31:393.; Miller et al., 1970, J. Bacteriol.104:1273) and has been very widely used in studies in E. coli and otherbacteria, and somewhat in fungi and animals. LAC fusions have been avery powerful tool due to the detailed genetic, biochemical andmolecular understanding of the operon and its encoded proteins and tothe availability of selective substrates such as lactose as well assubstrates for spectrophotometric, fluorometric and histochemicalassays. In addition, the wide availability of E. coli strains deficientin the components of the lac operon has enhanced its implementation inE. coli.

In spite of the remarkable success of lacZ fusions in the development ofE. coli molecular genetics, β-galactosidase fusions (Helmer et al.,1984, Bio/technology 2:520-527) are difficult if not impossible to useeffectively in plants because of very high endogenous β-galactosidaseactivity. β-Galactosidases are present in virtually all plants, and inmost, if not all tissues. They are also present in many bacteria andfungi of agricultural and biotechnological importance. Additionally,intrinsic β-galactoside compounds exist in all these organisms thatcould be degraded by the introduction of a β-galactosidase activity,hence altering the physiology of the organism. It is possible in atleast one plant system to selectively reduce or eliminate endogenousβ-galactosidase background activity under conditions for histochemicalanalysis leaving some bacterial β-galactosidase activity. However, thistreatment is not general, must be calibrated for each plant system,offers no quantitative methods, and hence is unlikely to opensignificant new prospects.

The Agrobacterium tumefaciens Ti-plasmid-encoded genes nopaline synthase(Depicker et al., 1983, J. Molec. Applied Genetics 1:561-575; Bevan etal., 1983a, Nature 304:184-187) and octopine synthase (De Greve et al.,1982, J. Mol. Applied Genetics, 1:499-513) have been used as reportergenes in the past because the opines produced by these enzymes are notnormally found in plant cells. Moreover the genes were readilyavailable, and are routinely transferred to plants upon Agrobacteriuminfection. However, these reporter genes are no longer widely usedbecause the assays are cumbersome, of limited specificity, difficult toquantitate (Otten et al., 1978, Biochem. Biophys. Res. Commun.527:497-500) and give no spatial information. In addition, octopinesynthase cannot tolerate amino-terminal fusions (Jones et al., 1985,EMBO J. 4:2411-2418).

Until recently, the two most widely used reporter genes have been thebacterial genes chloramphenicol acetyl transferase (CAT) and neomycinphophotransferase (NPTII) which encode enzymes with specificities notnormally found in plant tissues (Hererra-Estrella et al., 1983a, Nature303:209-213; Fraley et al., 1983, J. Histochem, Cytochem. 13:441-447).

CAT catalyzes the transfer of an acetate group from acetyl-coenzyme A toone or both of the free hydroxyl groups of the antibioticchloramphenicol, thus rendering it pharmacologically inactive. The geneand its encoded enzyme have been well characterized and the enzyme isquite stable. The most common assays involve incubation of an extractwith limiting concentrations of radioactively labelled chloramphenicoland excess acetyl CoA, followed by organic extraction of the reactionproducts, which are more hydrophobic than the substrate, separation ofthe products on thin layer chromatograms and resolution of theincorporated radioactivity by autoradiography. Quantitation of theradioactivity is then done by excision of the spots from the TLC andliquid scintillation counting. This is a relatively expensive andcumbersome assay (Gorman et al., 1982, Mol. Cell. Biol. 2:1044).Alternative assays have been developed using radiolabelled acetyl CoAthat avoid thin layer chromatography (Tomizawa, 1985, Cell 40:527-535;and Sleigh, 1986, Anal. Biochem, 156:251-256) but these are alsoexpensive and prone to difficulties in extrapolating from quantitationof incorporated acetate to enzyme concentrations. Recent developmentsusing HPLC, or fluorescently labeled chloramphenicol have streamlinedthis process somewhat.

NPTII, also called APH 3'II, catalyses the transfer of the terminalphosphate group from ATP to the antibiotic neomycin and its analogs,including geneticin (G418), and kanamycin. Chromatographic orelectrophoretic assays have been developed to detect the activity bymonitoring ³² P incorporation into the antibiotic after incubatingextracts with terminally ³² P labelled ATP and substrate (e.g., Reiss etal., 1984, Gene 30:217-223). NPTII can tolerate amino-terminal fusionsand remain enzymatically active, making it useful for studying transportinto organelles in plants.

Both CAT and NPTII are relatively difficult, tedious and expensive toassay and suffer from variable endogenous activities in plant and animalcells (generally caused by enzymes with broader substrate specificity),which limits their sensitivity. Competing reactions catalyzed byendogenous esterases, phosphatases, transferases and other enzymes alsomake quantitation of CAT or NPTII by enzyme kinetics very difficult, andquantitation without enzyme kinetics to be very suspect. In addition,there are no reasonable methods for identifying cell or tissuelocalization with these enzymes. Because NPT II is a very versatileantibiotic selection marker (an effector) for transformation of plants,it will probably continue to be used in this capacity for some time.However, its use as a reporter gene may be ephemeral.

Attention has recently focussed on methods for light production ingenetically engineered organisms using either of two luciferase genes.The firefly luciferase gene has been used as a marker in transgenicplants (Ow et al., 1986, Science 234:856-859), but the enzyme is labile,and is difficult and expensive to assay with accuracy (for a good reviewof the difficulties see DeLuca, M. and McElroy, W. D., 1978, Methods inEnzymology 57:3-15), the reaction is complex and there may be littlepotential for routine, affordable and meaningful histochemical analysisor fusion genetics. The genes luxA and luxB from Vibrio harveyi havealso been used in several studies to monitor gene action in transgenicplants (Koncz et al., 1987, Proc. Natl. Acad. Sci., U.S.A. 84:131-135;and Langridge et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:3219-323).However, detection of light production in situ by the expression of genefusions of luxA and luxB, or a fused luxAB requires rather sophisticatedsingle-photon capture and imaging systems may not unlikely to beaffordable in the foreseeable future to most laboratories. In addition,there are currently no available methods to alter the spectral output toovercome absorption by the sample.

Although there is the assertion, frequently made, that the firefly andbacterial luciferases offer the possibility of true in vivo analysis,these claims should be examined carefully. First both systems requirethe uptake of exogenous substrates into living cells--in the case of thefirefly luciferase, a complex charged molecule--in the case of thebacterial lux gene products, a relatively simple volatile aldehyde. Toproduce light, however, these compounds must then interact with theenzyme (modified and targeted to glyoxysomes in the case of the fireflyenzyme, a heterodimer of two gene products in the case of the bacterialluciferase) and with cofactors and other substrates: for fireflyluciferase Mg²⁺, O₂, ATP; for bacterial lux FMNH. The production oflight will therefore depend on high concentrations of not only theexogenously applied substrate (a difficult task to attain or measurereliably and reproducibly in all cell types) but also non-limitinglevels of the other co-factors and substrates. In fact, the assay forfirefly luciferase is so sensitive to ATP concentrations that it iswidely used to measure ATP levels in cells and extracts. While this isan interesting and important use of luciferase, it has only confoundingeffects on the use of luciferase as a gene fusion marker. Because of thecomplexity and expense of these systems and the increasingly apparentneed to quantitate gene action in situ, it is unlikely that the lightproduction methods will receive wide acceptance in the near future. Itmust always be recalled that the ultimate aim of quantitation, asregards gene fusion experiments, is not to quantitate photons or dyedeposition but rather gene activity.

3. SUMMARY OF THE INVENTION

The present invention relates to the β-glucuronidase (GUS) gene fusionsystem, and to the cloning and characterization of the β-glucuronidaseand glucuronide permease genes of Escherichia coli. It provides forrecombinant nucleic acid molecules which comprise β-glucuronidase andglucuronide permease encoding sequences, as well as functional portionsthereof.

In various embodiments of the invention, nucleic acid sequences encodingGUS may be combined with a second non-GUS encoding sequence to create aGUS gene fusion. For example, GUS encoding nucleic acid may be joined toa promoter/enhancer element so that the expression of GUS is controlledby the activity of the promoter/enhancer element. In specificembodiments of the invention, expression of GUS may be placed under thecontrol of an inducible, tissue-specific, or developmentally regulatedpromoter/enhancer element, including but not limited to the lacZpromoter of E. coli, the major sperm protein (msp-1) and col-1 promoterof Caenorhabditis elegans, or the ribulose bisphosphate carboxylase,cauliflower mosaic virus 35s, or patatin promoters, which are active inplants. According to various embodiments of the invention, GUS genefusion may be used to introduce GUS activity into a wide variety ofbacterial, microbial, plant, and animal host organisms. In furtherembodiments of the invention, GUS gene fusions may be used to provide anindex of gene activity in a host cell or organism, and thereby functionas a reporter gene system. In addition to its ability to withstand aminoterminal fusions, GUS offers the advantage over other reporter genes, inthat there is an almost complete absence of GUS activity in mostorganisms other than vertebrates and their attendant microflora.Surprisingly, lower and higher plants, bacteria, fungi, and many insectsproduce little or no GUS activity. A further advantage of the GUS genefusion system is the wide variety of available substrates, the ease andeconomy of GUS assay systems, and the fact that GUS activity may bedetected and accurately measured in single cells.

In further embodiments of the invention, GUS gene fusions may be used asan effective means of altering the cellular phenotype. For example, andnot by way of limitation, GUS gene fusions may be used to deliver abiologically active molecule to a specific target-cell or tissue. In aparticular embodiment, a biologically active molecule, such as acytotoxic substance (e.g. cycloheximide) may be coupled to D-glucuronicacid to form a nontoxic compound which will only be toxic to cells thatexpress GUS. If an organism that comprises a GUS gene fusion in whichGUS expression is controlled by a tissue-specific promoter is exposed tothis compound, tissues expressing GUS may be selectively ablated. Invarious embodiments of the invention, biologically active moleculesincluding growth hormones, sex hormones, cholesterol and antibiotics, toname but a few, may be targeted to GUS-expressing cells or tissues. Inanother specific embodiment of the invention, transgenic plantscomprising a GUS gene fusion are able to selectively benefit from theeffects of an auxin supplied in the form of a glucuronide.

In still further embodiments of the invention, nucleic acid sequencesencoding glucuronide permease may be used to introduce glucuronidepermease activity into a host cell or tissue, thereby rendering the hostpermeable to GUS substrates. The utilization of glucuronide permease inconjunction with the GUS gene fusion system broadens the potential usesto apply to virtually any organism and enables the detection of GUSactivity in vivo. Furthermore, by manipulating various characteristicsof permease activity, host cells may be engineered to be selectivelypermeable to certain GUS substrates but not others. The potential usesof the GUS gene fusion system in basic research, medicine, andagriculture are seemingly unlimited.

3.1. Abbreviations and Definitions

Chimetic gene: a sequence of DNA in which nucleotide sequences notnaturally occuring together are linked.

Gene fusion: A DNA construction (performed in vitro or in vivo) thatresults in the coding sequences from one gene (the "responder") beingtranscribed and/or translated under the direction of the controllingsequences of another gene (the "controller"). Responder genes can bedivided into two classes, reporters and effectors, with analytical Ormanipulative roles, respectively.

Translational fusions: gene fusions which encode a polypeptidecomprising coding information of the controller and responder genes.

Transcriptional fusions: gene fusions in which all coding sequences arederived from the responder gene.

4. DESCRIPTION OF THE FIGURES

FIG. 1 illustrates subcloning and strategy for determining thenucleotide sequence of the uidA gene.

FIG. 2 illustrates the DNA sequence of the bp insert of pRAJ220,containing the beta-glucuronidase gene.

FIG. 3 illustrates GUS gene module vectors.

FIG. 4 illustrates the results of 7.5% SDS-PAGE analysis ofbeta-glucuronidase. Aliquots of supernatants from induced and uninducedcultures of E. coli C600 were compared with aliquots of purifiedβ-glucuronidase. Lane (a) is molecular weight standards; lane (b) isextract from induced C600; lane (c) is extract from C600 induced forβ-glucuronidase with MeGlcU'; lane (d) is 0.3 μg of purifiedβ-glucuronidase; lane (e) is 3.0 μg aliquot of purified β-glucuronidase.

FIG. 5 illustrates structures of the col-l:GUS fusion (pRAJ321) and theMSP:GUS fusion (pRAJ421).

FIG. 6 shows the results of assaying beta-glucuronidase activity intransformed worms.

FIG. 7 illustrates the results of co-segregation analysis ofbeta-glucuronidase activity and transforming DNA; 40 F2 worms derivedfrom a transformant carrying the col-1:GUS fusion were cloned ontoindividual petri plates, grown to saturation, harvested and washed, andthe culture was split into two parts. Extracts were prepared for (a)fluorogenic beta-glucuronidase assays or (b) DNA dot blots--in the wellsof a Gilson tube rack.

FIG. 8 illustrates histochemical visualization of beta-glucuronidaseactivity in worms transformed with the col-1:GUS fusion (pRAJ321);

FIG. 9 illustrates the structure of expression vectors.

FIG. 10 is a graph illustrating beta-glucuronidase activity in extractsof different organs of transformed and non-transformed tobacco plants;extracts were prepared from old and young leaf, root, and stem fromrbcS-GUS (pBI131) and CaMV-GUS (pBI121) transformants of Nicotianatabacum, and then assayed for GUS activity by fluorometric assay, using4-methyl-umbelliferyl (4-mu) glucuronide as substrate, and measuring thefluorescence with excitation at 365 nm, emission at 455 nm to determinethe nanomolar (NM) concentration of fluorescent product.

FIG. 11 is an autoradiograph of a Southern blot of DNA extracted fromtransformed plants and digested with restriction endonucleases;

FIG. 12 illustrates 7.5% SDS-polyacrylamide gel stained forbeta-glucuronidase activity.

Lane 1. Transformed plant extract--CAB-GUS fusion.

Lane 2. Transformed plant extract--SSU-GUS 2.

Lane 3. Transformed plant extract--CaMV-GUS 21.

Lane 4. Non-transformed plant extract.

Lane 5. Non-transformed plant extract plus 1 ng GUS.

Lane 6. Non-transformed plant extract plus 10 ng GUS.

Lane 7. Non-transformed plant extract plus 50 ng GUS.

FIG. 13 illustrates the reaction catalyzed by β-glucuronidase, and thestructure of substrates transported by glucuronide permease;

FIG. 14 illustrates the structure of various plasmids. The subcloning ofthe 3' end of pRAJ210, encoding the glucuronide permease, was done bycleaving pRAJ210 with Pst I (cleaving in the polylinker site of pUC9,proximal to the Xho I site of pRAJ210 and Nsi I which cleaves just 5' ofthe BamHI site of pRAJ220. The sequence of this region was determined bythe method of Maxam and Gilbert;

FIG. 15 illustrates the DNA sequence of the glucuronide permease gene onpRAJ285. The sequence was determined as described in the legend to FIG.2. The sequence shown extends from the Nru I site within GUS (sequencedby dideoxy method) through the Nsi I site into the previouslyunsequenced region of pRAJ210. The sequence shown extends only just pastthe terminator codon of the permease. This is represented on the plasmidpRAJ285. The rest of pRAJ210 has been sequenced, and is present onpRAJ280-pRAJ284;

FIG. 16 is a comparison of the amino acid sequences of glucuronidepermease (top) and the melibiose permease (bottom) using the Universityof Wisconsin Genetics Computer Group Best-Fit programs. The linesbetween sequences indicate exact matches between the two sequences.Small gaps were introduced to maximize homology. Program parametersincluded: Gap Weight: 5.0; Average Match: 1.00; Length Weight: 0.30;Average Mismatch: -0.10; Quality: 41.8; Length: 9; Ratio: 0.098; Gaps:5; and

FIG. 17 is an analysis of the structure of the glucuronide permeaseusing the University of Wisconsin Genetics Computer Group PepPlotprogram. The bottom panel shows the hydropathy plot generated usingeither the Goldman or the Kyte and Doolittle criteria. The manyhydrophobic domains indicate potential alpha helical trans-membranesegments. This plot is very similar to a plot obtained analyzing themelibiose permease sequence.

FIG. 18. Effect of trypophyl glucuronide on GUS-expressing (column A)and control (column B) leaf discs on media containing 1) no auxin; 2) 1μM indole-3-acetic acid, 3) 1 μM tryptophyl-glucuronide, or 4) 10 μMtryptophyl glucuronide.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the β-glucuronidase gene fusion system,and to the cloning and characterization of the β-glucuronidase andglucuronide permease genes of E. coli. The invention provides for genefusions which comprise β-glucuronidase and/or glucuronide permease, fororganisms including plants, yeast and bacteria which comprise these genefusions, and for methods in which the GUS gene fusion system may be usedas a reporter gene or, alternatively, as a means of altering cellularphenotype.

For purposes of clarity of disclosure, and not by way of limitation, thedescription of the invention is divided into the following subsections:

(i) cloning and characterization of β-glucuronidase;

(ii) cloning and characterization of glucuronide permease;

(iii) Promoters that may be useful in the β-glucuronidase gene fusionsystem;

(iv) utility of β-glucuronidase gene fusions as reporter genes;

(v) utility of β-glucuronidase gene fusions in the manipulation ofcellular phenotype;

(vi) utility of glucuronide permease;

(vii) useful substrates for the β-glucuronidase gene fusion system; and

(viii) methods of analysis of β-glucuronidase expression.

5.1. Cloning and Characterization of Beta-Glucuronidase Genes

The present invention provides for recombinant DNA molecules whichencode biochemically active β-glucuronidase enzyme. In a specificembodiment of the invention, the recombinant DNA is derived from theEsherichia coli β-glucuronidase gene. In alternate embodiments of theinvention, the β-glucuronidase encoding nucleic acid is homolgous to theE. coli β-glucuronidase (GUS) gene, and/or may be derived from anotherorganism or species.

GUS-encoding nucleic acid clones may identified using any method knownin the art, including, but not limited to, the methods set forth inSection 6, infra. In particular, genomic DNA or cDNA libraries may bescreened for GUS-encoding sequences using techniques such as the methodset forth in Benton and Davis (1977, Science 196:180) for bacteriophagelibraries and Grunstein and Hogness (1975, Proc. Natl. Acad. Sci. U.S.A.72:396-3965) for plasmid libraries. Nucleic acid sequences known toencode GUS, such as, but not limited to, pRAJ210, described in Section6, infra, may be used as probes in these screening experiments.Alternately, oligonucleotide probes may be synthesized which correspondto nucleic acid sequences deduced from amino acid sequence of purifiedGUS enzyme.

The clones identified as containing sequences homologous to the GUS genemay then be tested for the ability to encode functional GUS enzyme. Forthis purpose, the clones may be subjected to sequence analysis, in orderto identify a suitable reading frame, initiation and terminationsignals. Alternatively, the cloned DNA sequence may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedprotein-coding sequence. A variety of host/vector systems may be used,including, but not limited to, bacterial systems (plasmid,bacteriophage, or cosmid expression vectors); mammalian cells infectedwith virus (e.g. vaccinia virus, adenovirus, etc.); insect cell systemsinfected with virus (e.g. baculovirus); yeast containing yeast vectors,etc. Appropriate promoter elements are listed in section 5.3, infra. Itis preferable to use an expression system in a host which exhibits lowintrinsic levels of GUS activity.

Expression systems transformed with GUS-encoding sequences may then beanalyzed for GUS activity using methods set forth in section 5.8, infra.GUS protein may be identified using anti-GUS antibodies, and wouldappear as a tetrameric protein with a monomer molecular weight ofapproximately 68-70 kDa.

The present invention provides for nucleic acid molecules which encodeGUS active enzyme, including, but not limited to, the nucleotidesequence substantially as depicted in FIG. 2, or portions thereof. Theinvention also provides for molecules homologous to the nucleic acidsequence depicted in FIG. 2, or a portion thereof, as defined byhybridization or sequence analysis. The invention also provides forgenetically altered forms of the GUS gene which may encode novel formsof GUS enzyme having a new or modified spectrum of substrate specificityand/or activity, as well as for GUS genes which would encode modifiedGUS protein, including, but not limited to, GUS linked to a signal ortransit peptide.

The functional equivalents of the nucleic acid sequences depicted inFIG. 2 are also provided for by the present invention. For example, thesequences depicted in FIG. 2 can be altered by substitutions, additionsor deletions that provide for functionally equivalent molecules. Forexample, due to the degeneracy of nucleotide coding sequences, other DNAsequences which encode substantially the same amino acid sequence asdepicted in FIG. 2 may be used in the practice of the present invention.These include but are not limited to nucleotide sequences comprising allor portions of the β-glucuronidase sequence depicted in FIG. 2 which arealtered by the substitution of different codons that encode the same ora functionally equivalent amino acid residue within the sequence, thusproducing a silent change. For example, one or more amino acid residueswithin the sequence can be substituted by another amino acid of asimilar polarity which acts as a functional equivalent. Substitutes foran amino acid within the sequence may be selected from other members ofthe class to which the amino acid belongs. For example, the non-polar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine, and histidine. The negatively charged (acidic)amino acids include aspartic and glutamic acid.

5.2. Cloning and Characterization of Glucuronide Permease Genes

The present invention provides for recombinant DNA molecules whichencode glucuronide permease protein. In a specific embodiment of theinvention, the recombinant DNA is derived from the E. coli glucuronidepermease gene. In alternate embodiments of the invention, theglucuronide permease may be derived from another organism or species.

Glucuronide permease encoding nucleic acid clones may be identifiedusing any method known in the art, including, but not limited to, themethods set forth in Section 9, infra. In particular, genomic DNA orcDNA libraries may be screened for glucuronide permease encodingsequences using methods such as that described by Benton and Davis(1977, Science 196:180) for bacteriophage libraries and Grunstein andHogness (1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961-3965) for plasmidlibraries. Nucleic acid sequences known to enclode glucuronide permease,such as, but not limited to pRAJ285, described in Section 9 infra, maybe used as probes in .these screening experiments. Alternatively,oligonucleotide probes may be synthesized which correspond to nucleicacid sequences deduced from amino acid sequence of purified glucuronidepermease protein.

The clones identified as containing sequences homologous to theglucuronide permease gene may then be tested for the ability to encodefunctional permease protein. Functional expression vectors may beconstructed as described for GUS in Section 5.1, supra. It may bepreferable to use the DNA containing permease sequence to transformcells which contain active GUS enzyme but which lack glucuronidepermease expression. For example, and not by way of limitation,bacterial cells which express, but do not secrete GUS, but lack afunctional glucuronide permease gene, when grown in the presence of acolorigenic GUS substrate, will not produce indicator color. If thesebacteria are transformed with DNA encoding glucuronide permease, andfunctional glucuronide permease is expressed, the colorigenic substratewill enter the cells, be cleaved by GUS, and release a colored indicatorsubstance. Any similar method of measuring glucuronide permease activitymay be used in which entrance of substrate into a cell is detectable,for example, the uptake of radiolabelled substrates, a method wellestablished in the art.

The present invention provides for nucleic acid molecules which encodeglucuronide permease, including, but not limited to, the nucleotidesequence substantially as depicted in FIG. 15, or portions thereof. Theinvention also provides for molecules homologous to the nucleic acidsequence depicted in FIG. 15 or a portion thereof as defined byhybridization or sequence analysis. The sequence similarity to themelibiose permease, which has a substrate specificity which differs fromglucuronide permease, would indicate that a variety of permeasemolecules homologous to the glucuronide permease are likely to exist.The invention also provides for genetically altered forms of glucuronidepermease which may be more or less selective in the choice of moleculeswhich may be transported into the cell, or otherwise altered in itsactivity. Further, the invention provides for functional equivalents ofthe nucleic acid sequences depicted in FIG. 15, as discussed in Section.5.1 relative to β-glucuronidase.

5.3. Promoters That May be Useful in the Beta-glucuronidase Gene FusionSystem

According to the invention, expression of GUS or glucuronide permeasemay be controlled by any known promoter/enhancer element known in theart. Promoters which may be used to control expression include, but arenot limited to, the SV40 early promoter region (Bernoist and Chambon,1981, Nature 290:304-310), the promoter contained in the 3' longterminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78:144-1445), the regulatory sequences ofthe metallothionine gene (Brinster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the β-glactamase promoter(Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25), see also "Useful proteins from recombinantbacteria" in Scientific American, 1980, 242:74-94; plant expressionvectors comprising the nopaline synthetase promoter region(Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaicvirus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871),and the promoter for the photosynthetic enzyme ribulose biphosphatecarboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120);promoter elements from yeast or other fungi such as the Gal 4 promoter,the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)promoter, alkaline phophatase promoter, and the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells (Swift et al., 1984, Cell38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene controlregion which is active in pancreatic beta cells (Hanahan, 1985, Nature315:115-122), immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58);alpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al, 1987, Genes and Devel. 1:161-171), beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378). Furthermore, promoter/enhancer elements which are activein plants may also be utilized, including, but not limited to,light-responsive promoter sequences such as ribulose bisphosphatecarboxylase (Coruzzi et al., 1984, EMBO J. 3: 1671-1680;Herrera-Estrella et al., 1984, Nature 310:115-120), the chlorophyll a/bbinding protein (Cab) of the light-harvesting chlorophyll-proteincomplex (Apel et al., 1978, Eur. J. Biochem. 85: 581-588; Stiekema etal., 1983, Plant Physiol. 7.2:717-724; Thompson et al., 1983, Plants158: 487-500; Jones et al., 1985, EMBO J. 4: 2411-2418) and the ST-LS1gene of potato (Stockhaus et al., 1989, Plant Cell 1: 805-814).Additional plant promoter sequences which may be used include but arenot limited to the soybean heat shock protein hsp17.5-E or hsp17.3-Bpromoters (Gurley et all, 1986, Mol. Cell Biol. 6: 559-565); theParasponia andersoni hemoglobin promoter (Landsmann et al., 1988, Mol.Gen. Genet. 214:68-73); the phenylalanine ammonia-lyase promoter, whichappears to be active in specific cell types which accumulatephenyl-propanoid derivatives in response to wounding and also duringnormal development of the xylem and flower (Bevan et al., 1989, EMBO J.8:1899-1906); and the petunia 5-enolpyruvylshikimate-3-phosphatesynthase gene promoter (Benfey and Chua, 1989, Science 244:174-181)including the Rhizobium meliloti FIXD gene promoter described in U.S.Pat. No. 4,782,022, issued Nov. 1, 1988, by Puhler et al.; the nopalinesynthase promoter (Ha and An, 1989, Nucleic Acids Res. 17:215-224; An etal., 1988, Plant Physiol. 88:547-552); rol A, B and C promoters ofAgrobacterium rhizogenes (Schmulling et al., 1989, Plant Cell 1:665-670;Sugaya et al., 1989, Plant Cell Physiol. 30:649-654); the patatinpromoter (Rocha-Sosa et al., 1989, EMBO J. 8:23-29) and the cauliflowermosaic virus (CaMV) 35S promoter (Odell et all, 1985, Nature313:810-812; Jensen et al., 1986, Nature 321:669-674; Jefferson et al.,1987, EMBO J. 6:3901-3907; Kay et al., 1987, Science 236:1299-1302; andSanders et al., 1987, Natl. Acids Res. 14:1543-1558).

5.3.1. Introduction of Beta-Glucuronidase and/or Permease into a HostCell or Organism

Recombinant DNA comprising nucleic acid sequence encoding GUS orglucuronide permease, together with an appropriate controller sequence,may be introduced into a host cell or organism,using any method known inthe art, including, but not limited to, transfection, transformation,infection, or microinjection, utilizing methods including, but notlimited to, calcium phosphate or DEAE-dextran transformation,electroporation, or cell gun.

In preferred embodiments of the invention, the Agrobacterium tumefaciensgene transfer system may be used to introduce the recombinant constructsof the invention into plants; generally, this system may be utilized totransfer DNA into dicotyledonous plants (Bevan et al., 1982, Ann. Rev.Genet. 16:357-384; Rogers et al., 1986, Methods Enzymol. 118:627-641;Fraley et al., 1986, CRC Crit. Rev. Plant Sci. 4:1-46; Hooykaas et al.,1984, Adv. Genet. 22:210-283; Nester et al., 1984, Ann. Rev. PlantPhysiol. 35:387-413). To this purpose, vectors such as, but not limitedto, binary Agrobacterium vectors for plant transformation may beutilized, such as, for example, the vector described by Bevan (1984,Nucl. Acids Res. 12:8711-8721). Xanthi may be transformed by a leafinoculation procedure such as that described by Horsch et al. (1985,Science 227:1229-1231).

Additional methods for introducing DNA into plants may also be utilized,particularly if the recombinant construct is to be used to create atransgenic monocotyledonous plant. Such methods would include, but arenot limited to poly(ethylene glycol) and calcium-mediated uptake ofnaked DNA (Hain et al., 1985, Mol. Gen. Genet. 199:161-168; Paszkowskiet al., 1984, EMBO J. 3:2717-2722; Potrykus et al., 1985, Mol. Gen.Genet. 199:169-177), electroporation (Fromm et al., 1985, Proc. Natl.Acad. Sci. U.S.A. 82:5824-5828), microinjection, macroinjection andparticle bombardment.

5.4. Utility of Beta-Glucuridase Gene Fusions as Reporter Genes

According to the invention, GUS gene fusions may be utilized as areporter gene system. Advantages of using the GUS gene fusion systeminclude the persistent activity of GUS despite amino-terminal fusions,the abundant substrates for GUS available, and the ease and economy ofmany GUS assays. Furthermore, GUS activity may be detected in samples assmall as a single cell.

In various embodiments of the invention, GUS gene fusions may be used astranscriptional gene fusions or translational gene fusions.

According to particular embodiments of the invention, GUS gene fusionscan be used to measure the activity of a controller element. Forexample, and not by way of limitation, a gene fusion comprising a GUSencoding nucleic acid under the control of a promoter/enhancer element,X, could be used to generate a transgenic plant. Tissue-specificactivity of promoter X would be detectable by the observation that GUSactivity was expressed in some plant tissues, but not others. Similarly,a GUS gene fusion comprising controller sequences such as a ribosomebinding site or another translation-related sequence, a signal peptide,or a chloroplast or nucleus-target peptide, to name but a few, could beused to test the activities and explore the properties of the controllerelement.

According to further embodiments of the invention, GUS gene fusionscould be used to report on the expression of a second gene of interest.In this context, gene of interest is construed to mean any gene whichencodes a product (e.g. RNA or protein) that is of interest, i.e., thatis the subject of study or design. For example, and not by way oflimitation, a particular gene of interest, Y, improves tobacco cropyield. GUS gene fusions may be used in various ways to report on whetheror not gene Y is expressed, including, but not limited to, thefollowing. First, a recombinant construct which contains a promoterelement which will result in expression appropriate for gene Y (withregard to developmental timing, tissue specificity, etc.) may be placedon the same construct as the GUS gene; expression of GUS activity shouldbe controlled by the same promoter sequence which controls expression ofY, but may or may not be transcribed on the same RNA molecule.Downstream of Y, and separated from Y by a translational stop signal, isthe GUS gene. This construct is used to create a transgenic tobaccoplant. It would be impractical and overly time consuming to produce acrop of plants in order to determine whether Y were expressed or not.Instead, it would be straightforward to identify transgenic plants whichmay express Y by testing for GUS activity. As part of a singletranscription unit with Y, GUS may only be transcribed if Y has beentranscribed. Second, two constructs, one comprising Y and itsappropriate promoter element, the second comprising GUS and the samepromoter, may be inserted into a transgenic tobacco plantsimultaneously. To identify plants which express Y, one may identifyplants that express GUS, as the expression of both genes is under thecontrol of the same promoter.

According to related embodiments, GUS gene fusions may be used to assaymutagenic potential. For example, GUS encoding sequence or a controllersequence could be engineered such that GUS would not be expressed incells that contain the engineered gene fusion. If these cells weresubjected to a mutagen, reversion of the mutation could restore GUSactivity. Similar experiments could be used to study cis and transrelationships between controller elements. Likewise, inactivation of GUSexpression could also be used to assay mutagenic activity. According toa further embodiment, GUS expression may be inactivated by transposoninsertion; upon excision, GUS gene expression would be activatedresulting in cells that would be "marked" by GUS activity. Conversely,GUS-encoding sequences could be incorporated into a transposableelement; relocation of this element in the genome may be observed toeffect activation of GUS expression.

The virtual lack of GUS activity in many bacteria, plants and insectsrenders GUS a particularly useful reporter enzyme. Furthermore, itappears that GUS itself does not significantly alter the physiology ofhost cell transformants, rendering it suitable for in vivo use. In theExample sections that follow, the successful use of GUS gene fusions isexemplified by expression of active GUS enzyme directed by distinctpromoter elements in organisms as diverse as bacteria, nematodes, andhigher plants.

In further embodiments of the invention, GUS gene fusions may be used toconfer a selectable phenotype on cells. According to the current stateof the art, when a collection of cells is subjected to routine DNAtransfer methods, it is important to be able to select for further studythose cells which have successfully incorporated the exogenous DNA.Standard methods frequently involve the co-transfer of genes forantibiotic resistance, or resistance to another compound whichordinarily would be toxic to cells. Exposure to antibiotic or toxinshould kill cells that have not been successfully transformed, whereascells containing and expressing transferred DNA sequences shouldsurvive. It has been considered, however, whether the selection agenthas in fact resulted in the survival of a subpopulation of cells whichnot only contain express transferred DNA but which also exhibitadditional phenotypic differences from the original host cell. In orderto preclude this possibility, a method of selection which identifiestransformants solely on the basis of expressed transferred DNA sequencesmay be utilized. For example, potential transformants, comprising a GUSgene fusion, may be tested for expression of GUS activity; of note, GUSmay be identified within single cells. In a specific embodiment of theinvention, a gene fusion may be constructed in which a GUS gene, underthe control of a suitable promoter, is linked to a signal peptide; thisgene fusion is used to transform cells. If expressed, GUS may besecreted from the cell via the signal peptide, and may be detected insupernatants or cloned transformants.

5.5. Utility of Beta-Glucuronidase Gene Fusions in the Manipulation ofCellular Phenotype

According to the invention, GUS gene fusions may be used to manipulatecellular or organismal phenotype. In various embodiments of theinvention, a β-glucuronide comprising a bioactive compound enters a cellwhich comprises a GUS gene fusion and is cleaved by GUS enzyme such thatthe bioactive compound is released and is capable of acting on the cell.The term "bioactive compound" is construed to refer to any compoundwhich has any effect, including inductive as well as inhibitory effects,on a cell or organism, and refers to compounds including, but notlimited to, growth factors, differentiation factors, hormones,antibiotics, steroid compounds, toxins, lymphokines, etc. In preferredembodiments of the invention, the bioactive compound is inactive whencomprised in the glucuronide.

In various embodiments, GUS gene fusions may be used to manipulatecellular, or organism, phenotype through the action of exogenouslysupplied glucuronides. For example, it may be desirable to make aparticular cell population, tissue, or organism selectively susceptibleto a particular bioactive compound. Susceptibility may be conferred (i)by the presence of GUS enzyme, where, in nature, no GUS activity isfound; (ii) by increased levels of GUS enzyme; (iii) by the presence ofan altered form of GUS which is particularly active toward glucuronidescomprising the bioactive compound of interest; or (iv) by increaseduptake of substrate.

As an example, and not by way of limitation, it may be desirable topromote the growth of crop plants, but not the growth of weeds.Transgenic crop plants may be created which comprise a GUS gene fusionin which GUS expression is controlled by a powerful promoter.Preferably, these plants are also rendered permeable to glucuronides byexpression of a transgene which encodes glucuronide permease. If theseplants are sprayed with a glucuronide which comprises an auxin, theconjugated auxin will be freed by GUS activity within the plant cells,where it might subsequently act to promote plant growth. Because weedslack endogenous GUS activity, they would be unaffected by conjugatedauxin. According to a specific embodiment of the invention, exemplifiedin Section 10, infra transgenic plants comprising a CaMV 35 S/GUS genefusion are selectively able to metabolize auxin supplied in the form ofa glucuronide derivative.

As another example, the invention may be utilized to ablate a specifictissue or population of cells. According to particular embodiments ofthe invention, gene fusions may comprise GUS under the control of Z, atissue specific promoter element. Accordingly, recombinant GUS shouldonly be expressed in tissues in which promoter Z is active. If atransgenic organism comprising the Z-GUS gene fusion is exposed toglucuronide comprising a toxin, the toxin would only be activated bytissues expressing Z-GUS. In specific embodiments of the invention, thistechnique may be used to create male-sterile plants, for instance, byensuring that Z-GUS expressed GUS activity only in anthers and pollens,or, alternatively, animal models for organ degeneration, to name onlytwo of the numerous applications.

In related embodiments, GUS gene fusions may be constructed such thatGUS is only expressed after a particular event has occurred. Forexample, a gene fusion may be constructed in which GUS is expressedunder the control of a promoter which responds to a trans-activatingfactor. In specific embodiments, this trans-activating factor may be aviral transactivating factor, such that the only cells that wouldexpress recombinant GUS would be virus-infected cells. Accordingly,virus-infected cells would be particularly sensitive to glucuronidecomprising an antiviral or cytotoxic agent.

Importantly, this technology may not only be applied to the treatment ofviral infections in organisms which lack endogenous GUS activity, suchas plants, but also organisms which do possess GUS activity, because GUSlevels can be elevated to levels which exceed normal levels (therebyconferring a selective sensitivity to GUS substrates) or, alternatively,because recombinant GUS may be engineered to possess a higher or moreselective activity. Selectivity may also be conferred by recombinantglucuronide permease, as discussed infra.

In further embodiments of the invention, GUS gene fusions may be used tomanipulate cellular, or organism phenotype through the action ofendogeneusly generated glucuronides. For example, in organisms, such asvertebrates, which metabolize a number of compounds including hormones,non-hormonal steroids (including cholesterol) and antibiotics toinactive glucuronides, GUS gene fusions may be used to alter thebioavailability of these compounds. By either augmenting endogenouslevels of GUS or providing altered selectivity of the GUS enzyme orglucuronide permease, the gene fusions of the invention may increase ordecrease the half-life of a number of biologically significantmolecules.

For example, contraception may be a desirable result of increased levelsof estrogen and/or progesterone, both of which are excreted viaconjugation to form glucuronides. A transgenic animal comprising a GUSgene fusion transgene may express either (i) higher levels of GUS thanwould normally be expressed in the animal; (ii) an altered form of GUSwhich would be particularly effective at hydrolizing theestrogen/progesterone-glucuronide linkage; (iii) a form of glucuronidepermease exceptionally adept at bringing estrogen/progesteroneglucuronides into the cell; or (iv) a compartmentalized form of GUS.Endogenously produced estrogen/progesterone would thus have a longerhalf-life in these animals and contraception could be achieved withoutany exogenously administered hormone. This technology may be applied todomestic animals, such as dogs and cats. Because increased levels ofestrogen or progesterone may have deleterious effects on nonreproductiveorgans, GUS gene fusions may desirably be expressed only in reproductivetissues, thus increasing GUS activity in estrogen/progesteone targetorgans at the cellular level without altering serum levels in theorganism. In this embodiment of the invention, contraception could bepermanent (unless, for example, GUS expression were controlled by aninducible promoter). Alternatively, hormonal levels could be manipulatedto increase fecundity, as in domestic livestock. Importantly, the GUSgene fusion system may be used to temporarily alter the bioavailabilityof compounds endogenously incorporated into glucuronides.

Temporary alteration of glucuronide metabolism may be achieved byexploiting the symbiotic relationship between vertebrates and theirintestinal flora. Typically, bioactive compounds are inactivated in theliver by glucuronidation. The resulting glucuronides are secreted intothe bile ducts and then into the intestine. Intestinal bacteria useendogenous GUS to utilize glucuronides as a carbon source, and therebyrelease significant amount of deconjugated bioactive substance whichrecycles into the bloodstream. In various embodiments of the invention,the levels, activity, and specificity of GUS or alternatively ofglucuronide permease in the intestinal bacteria may be altered in orderto change the rate of recycling of bioactive compound. For example, andnot by way of limitation, bacteria may be produced which comprise a GUSgene fusion which results in extremely high levels of GUS activity inthe bacteria or GUS activity with high specific activity. If avertebrate's intestine is populated with bacteria which express highlevels of GUS, bioactive compounds will be recycled at a higher rate andthe half-life of bioactive compounds excreted via the glucuronidepathway will be extended. In this way, the half-life of antibiotics maybe extended, thereby increasing the period between doses, or temporarycontraception may be achieved via increased estrogen or progesteronelevels to list but two of numerous examples. Alternatively, bacteria maybe engineered which have lower GUS or glucuronide permease activity,thereby decreasing recycling of substances which are desirably excretedfor example, ingested toxins, mutagens, etc. For example, patients withhigh cholesterol levels may benefit from an intestinal population ofbacteria which have low levels of GUS activity and which permit theexcretion of cholesterol as a glucuronide.

As discussed above, by using GUS enzyme or permease which is substrateselective, in particular embodiments of the invention the recycling ofsome compounds, but not others, may be altered. In preferred embodimentsof the invention, bacteria comprising a GUS gene fusion further comprisea gene for resistance to a particular antibiotic; in this manner, theantibiotic may be used to eliminate the vertebrate's natural bacteriaflora, and permit population by recombinant bacteria. Recombinantbacteria, in turn, may be eliminated by the use of a second antibioticto which they carry no resistance.

It should be understood that the abovementioned examples are notlimiting to the invention, but are intended only to illustrate a few ofthe numerous embodiments of the invention. The invention furtherprovides for variations of these embodiments which may become apparentto one skilled in the art after reading the examples contained herein.

5.6. Utility of Glucuronide Permease

According to the invention, glucuronide permease encoding nucleic acids,under the control of a suitable promoter element, may be inserted intocells to render the cells permeable to glucuronide substrates of GUSenzyme.

The glucuronide permease gene may be used in many different organisms totransport substrates for β-glucuronidase (GUS) into cells. Because thispermease activity is encoded by a single polypeptide, and because thereis no subsequent modification of the permease required for its insertioninto membranes or its function (by analogy with the melibiose andlactose permeases) it is reasonable to expect that expression of thepermease under the control of virtually any promoter in a transgenicorganism will result in the transport of β-glucuronides into cells ofthat organism.

In one embodiment of the present invention, the glucuronide permeasegene can be transfected together with GUS as part of the same construct,or incorporated into another vector (i.e. cotransfector), such as aplasmid or a eukaryotic vector such as SV-40 (Mulligan and Berg, 1980,Science 209:1422-1427). This in turn will allow for β-glucuronidase,including fluorogenic and colorimetric β-glucuronidase substrates, to beincorporated into live, undisrupted cells, thus allowing detection ofβ-glucuronidase reporter gene activity in vivo, eliminating theconstraints of tissue extracts and histologic procedures, and therebyproviding for more general applicability of the GUS system.

In another embodiment of the present invention, the glucuronide permeasegene can be introduced into cells which have endogenous β-glucuronidaseactivity. By altering the number of glucuronide permease moleculespresent at the cell membrane, permeability of the membrane toβ-glucuronidase can be effected, and thus, glucuronide permease itselfcan function as a reporter gene and allow the metabolism of inactiveglucuronides to active aglycones using endogenous β-glucuronidase. Thiscould be applied not only to cells, but to whole organisms, to produce,for example, glucuronide permease plants (see infra).

Glucuronide permease can be produced in large amounts by inserting thegene into an active expression vector and allowing the gene to beexpressed, for example, in bacteria. In one embodiment of the presentinvention, the glucuronide permease could be chemically or geneticallylinked to a ligand, which could deliver the permease for insertion intocell membranes bearing the ligand receptor. By analogy to lactose andmelibiose permease, the glucuronide permease should be able to integratespontaneously into the cell membrane. For example, glucuronide permeasecould be coupled to the Fc region of an immunoglobulin specific for adiscrete population of mammalian cells. Upon binding to these cells, theantibody would deliver glucuronide permease for insertion into the cellmembrane, thereby making a discrete population of mammalian cellsincreasingly permeable to β-glucuronides. In this example, selectedβ-glucuronides could then be used to various purposes (see infra),utilizing endogenous β-glucuronidase expressed by mammalian cells.

In another, related embodiment of the present invention, glucuronidepermease could be incorporated into membrane vesicles, and therebybecome inserted into the cell membrane. The membrane vesicles couldcontain various substances, including, but not limited to, β-glucuronideconjugated compounds.

The present invention provides for the use of glucuronide permease inaltering membrane permeability to various compounds, utilizingβ-glucuronide conjugates and endogenous or exogenously-suppliedβ-glucuronidase activity. These compounds include, but are not limitedto, the following substances which may be conjugated to β-glucuronicacid to form β-glucuronides, which may be transported by glucuronidepermease.

(a) Indicator substances such as histochemical indicators includingnapthol and napthol ASB1 and X-gluc (supra); fluorogenic substances suchas 4-methyl umbelliferone and fluoroscein 3-0-methylfluoroscein, or4-trifluromethyl umbelliferon and colormetric indicators such asresorufin, and p-nitrophenol.

(b) Catabolic substances such as cellobiuronic acid, a disaccharidewhich, when transported into the cell, is metabolized to glucose byβ-glucuronidase.

(c) Growth factors, such as the various peptide growth hormones, and inplants, cytokinins or auxins, gibberellins or abscisic acid.

(d) Toxic substances, such as snake venom toxins, including, accordingto their mode of action, cardiotoxins which cause irreversibledepolarization of the cell membranes of heart muscles or nerve cells,neurotoxins which prevent neuormuscular transmission by blockingneurotransmitter receptors, and protease inhibitors which inhibitacetylcholine esterase and similar enzymes involved in nervetransmission. Also included are phytotoxins such as ricin and abrin andbacterial toxins, fungal toxins, herbicides such as dinitrophenolderivatives, or sulfonyl ureas, etc. and chemotherapeutic agents.

(e) Various steriod hormones are excreted as conjugated β-glucuronides.Usong glucuronide permease, these hormones could be recycled, and theirmechanism of action potentiated in select cells. In another embodimentof the present invention, exogenous steroid-β-glucuronides could betargeted to glucuronide permease expressing cells.

5.7. Additional Uses of the GUS System

In addition to the embodiments described above, the present inventionalso envisions the utilization of further aspects of glucuronidemetabolism in molecular engineering of organisms. For example, therepressor encoded by the GUS operon may be utilized as a means ofturning on or turning off of GUS activity in recombinant organisms, andmay also be useful in conferring selectively to GUS activity towardparticular substrates. Furthermore, because plants appear to lack anendogenous GUS/glucuronidation system, it is envisioned that transgenicplants may be created which express glucuronidyl transferases which maybe capable of conjugating compounds including, but not limited to,herbicides, to form inactive glucuronides; such plants may beconstitutively resistant to toxins such as herbicides.

Therefore, according to the invention, the repressor of β-glucuronidaseexpression, which has been mapped upstream of the structural gene forGUS, may be cloned by using standard techniques to "walk" upstream ofthe GUS structural gene, and should be identifiable as an open readingframe. The repressor may be cloned and sequenced using standardtechniques, expressed in an expression vector using techniques discussedin Sections 5.2 and 5.3, supra, and then tested for the ability torepress expression of GUS, for example, in bacteria which constitutivelyexpress GUS. Alternatively, the binding site for the repressor may beidentified (for example, by footprinting analysis), and may thereby beused to repress GUS expression.

5.8. Useful Substrates for the Beta-Glucuronidase Gene Fusion System

Virtually any β-D-glucuronide may be used as a substrate according tothe invention. A variety of glucuronides may be used in GUS assaysystems, including, but not limited to, fluorescent glucuronides such as4-trifluoromethyl umbelliferyl β-D-glucuronide, 3-cyanoumbelliferylβ-D-glucuronide, and fluoroscein β-D-glucuronide; and chromogenicsubstrates such as 5-bromo-4-chloro-3-indoyl glucuronide, and naphtholASBI-glucuronide, cleaved to liberate free naphthol ASBI, then coupledto a diazo dye. As discussed supra, glucuronides comprising bioactivemolecules can also be used as GUS substrates according to the invention;useful bioactive compounds include, but are not limited to, steroidhormones non-steroid hormones and factors, lymphokines, auxins,cytokinins, giberellins, toxins, vitamins, cofactors and antibiotics toname but a few.

5.9. Methods of Analysis of GUS Expression

5.9.1. Lysis and Extraction

Cells, tissues, cultures or whole organisms can be homogenized forassays in a variety of different buffers, using any method known in theart. The method of lysis and extraction may depend on the nature of thesample. Useful methods include but are not limited to the French pressor sonicator for bacterial and yeast samples, and grinding with sandusing a mortar and pestle for plant tissues. However, almost any othermethod of homogenization can be seriously considered. Small disposablepestles that fit into Eppendorf tubes (Kontes Glass) are suitable forhomogenizing small pieces of tissue (e.g. leaf tissue), but areinadequate for fibrous tissue such as root and stem, or fungal andbacterial cultures. Repeated freeze-thaw cycles can be very effective atbreaking open many tissues and cells, but must be approached cautiouslyfor use in reproducibly extracting maximal enzyme activity.

5.9.2. Composition of Extraction Buffers

Any number of different extraction buffers may be utilized; preferably,a buffer system should be devised to address some of the characteristicsof the GUS enzyme and the properties of extracts from the host organism.One extraction buffer that has been found to work well for plant, fungaland bacterial studies is described in Table I. The rationale for thisparticular extraction buffer is set out below.

                  TABLE I                                                         ______________________________________                                        GUS Extraction Buffer (11)                                                                    Stock Solutions Volumes                                       ______________________________________                                        50 mM NaHPO.sub.4, pH 7.0                                                                     1M NaHPO.sub.4, pH 7.0                                                                        50 ml                                         5 mM dithiothreitol (DTT)                                                                     1M DTT in H.sub.2 O                                                                            5 ml                                         1 mM Na.sub.2 EDTA                                                                            0.5M Na.sub.2 EDTA, pH 8.0                                                                     2 ml                                         0.1% Sodium Lauryl                                                                            10% Sarcosyl    10 ml                                         Sarcosine                                                                     0.1% Triton X-100                                                                             10% Triton      10 ml                                         H.sub.2 O                       923 ml                                        ______________________________________                                    

The phosphate is present to maintain the pH at or around neutrality,where GUS is fully active and stable, and as an ionic contribution.Other buffers are likely to function at least as well, with theadvantage of not precipitating Ca²⁺ from added media. Tris and HEPES donot appear to affect GUS activity adversely, although Tris has a verypoor buffering capacity in the pH range of maximal GUS activity(5.5-7.5).

Dithiotreitol may be added to help maintain the sulfhydryl groups of GUSin a reduced state. This may be important to achieve and maintainmaximum GUS activity. Higher concentrations also function very well, asdoes β-mercaptoethanol at 10-100 mM concentrations. β-mercaptoethanol isnot as strong a reducing reagent as DTT, and because of its volatilitymay not be preferred. Some plant extracts are highly oxidizing, and soincubation with excessive β-mercaptoethanol or DTT on ice afterextraction may help to reactivate any GUS that has become reversiblyoxidized. It has occasionally been observed that assays of freshlyprepared extracts may give a somewhat lower GUS activity that does notalways show linear kinetics. This may be due to the time-dependence ofsulfhydryl reactivation by reducing agents. This lag is longer with lowconcentrations of β-mercaptoethanol and progressively shorter withhigher concentrations, or with stronger reducing agents, such as DTT.Inclusion of DTT at 5 mM or higher usually results in full activityalmost instantaneously upon extraction.

GUS does not appear to require any ionic cofactors, but it is inhibitedto various degrees by certain divalent metal cations. Accordingly, itmay be preferable to include EDTA as a prophylactic measure to chelatethese ions. Additionally, various oxidation reactions in plant extracts,which may cause browning and accumulation of other colored andinhibitory substances, require divalent cations, and may be inhibited byEDTA. Higher concentrations are not found to be inhibitory to GUS. Forsubsequent analysis of DNA concentration in the extract by fluorogenicmethods (Labarca, C. and Paigen, K., 1980, Anal. Biochem. 102:344-352;Jefferson et al., 1987, supra), it is preferable to maintain an adequateconcentration of EDTA to chelate Mg²⁺ ions required for DNAse action, sothat the integrity of the DNA may be preserved.

The detergents Triton X-100 and Sarcosyl may be included to increase theefficiency of extraction by helping to lyse cells and subcellularorganelles, and to prevent aggregation of the enzyme. Triton effectivelylyses organelles such as chlorplasts (e.g. Kavanagh et al., 1988, Molec.Gen. Genet. 215:38-45). Nuclei are efficiently lysed by Sarcosyl but notby Triton, so if a DNA measurement is planned, inclusion of Sarcosyl isrecommended. GUS does not appear to be adversely affected by thesedetergents in modest concentrations. SDS at low concentrations alsoappears to be tolerated. Triton X-100 has been reported to have anadverse effect on the subsequent assay of GUS using resorufinβ-glucuronide by contributing an endogenous fluorescence at long wavelengths.

5.9.3. Protease Action on GUS

GUS is remarkably resistant to protease action, with a very longhalf-life in living cells and in most extracts, but if proteases are apotential problem, due to unusual circumstances such as proteolyticallysensitive gene fusions, PMSF (phenylmethyl sulfonyl fluoride--a potentinhibitor of serine proteases) at a final concentration of at least 2 mMmay be included in the extraction buffer. Other proteinase inhibitorssuch as leupeptin (1 mM) and aprotinin (100 μg/ml) have also been usedsuccessfully (Kavanagh et al., 1988, supra). Substantially moreproteinase K appears to be required to destroy GUS activity in vitrothan seems to be necessary for many other proteins.

5.9.4. Storage of Extracts

Extracts may be quickly frozen in liquid nitrogen and stored at -70° C.,with no loss of activity for a long time (a year at least), and at 4° C.with very little loss. Slow freezing to -70° C. may be adequate forstorage, but evidence suggests caution in this mode of freezing. It ispreferable that initial experiments on test samples be carried outbefore committing to a particular method of storage. Avoid storage at-20° which appears to cause a rapid decrease in enzyme activity.However, tissues stored at -20° C. for a few days do not seem to losesignificant GUS activity.

5.9.5. Treatment of Extracts to Reduce Endogenous Fluorescence ofAbsorption

Tissues or cells that are high in endogenous light-absorbing orfluorescent compounds or that produce high levels of polyphenolics thatmight inhibit subsequent analysis can be extracted in GUS extractionbuffer with polyclar AT (insoluble polyvinvyl pyrollidone) which adsorbspolypphenols and ligines, followed by a brief spin-column of SepharoseCL6B, Sephadex G-25, or a comparable resin to eliminate almost allpolyphenolics and low-molecular weight fluorescent contaminants from theextract. The routine use of spin-columns may be extended to microtiterplate spin columns as well, to process large numbers of extracts.

5.9.6. β-Glucuronidase Assays

Detection of β-glucuronidase activity, like all enzyme measurements,depends on the availability of substrates for the enzyme which, whenacted on by the enzyme, liberate a product that is distinguishable fromthe substrate. A substrate that is designed to maximize sensitivity ofdetection of the enzyme should, preferably, have several characteristicproperties. Optimally, there should be a method of detecting the productvery specifically; the substrate should be cleaved only by the enzymeunder study, with minimal spontaneous cleavage, and the signal to noiseratio of the method of detection should be as high as possible. Inaddition, one should consider whether quantitation of the signal will beimportant, and whether a direct relationship between product formationand enzyme activity will exist under the conditions of assay.

Quantitative enzyme assays may preferably be done in extracts, where theconditions of assay can be carefully controlled, and optimized to givereliable and accurate results. However, in working with multicellularorganisms, where it is becoming increasingly apparent that differencesin gene action between neighboring cells must be resolved, it is alsoessential to have qualitative enzyme assay methods in which the productis localized to the site of activity. This will therefore indicate thecells/tissues/colonies in which GUS-and presumably the GUS genefusion--is active, and allow discrimination between neighboring sitesthat differ in their activity. Ideally, spatial localization andrestriction of product should also be quantitative.

Quantitative measurements of GUS activity performed in extracts may useabsorption or fluorescence methods. Although the possibility exists forfluorescent methods for GUS that will localize the fluorescent productat the site of enzyme activity, all the currently available methods forGUS histochemistry employ light absorption methods (color deposition).

5.9.6.1. Fluorogenic Assays

Detection of fluorescent molecules offers a very high signal-to-noiseratio because the incident excitation light does not impinge on thedetection apparatus, and has a spectrum distinct and separable from thatof the emission. The use of fluorescence measurements to detect enzymeactivity usually gives two to four orders of magnitude (100-10,000×)greater sensitivity than methods that rely on spectrophotometricdetermination of product concentration by absorption. Absorptiontechniques measure a small difference between two large values, whereasfluorescence techniques measure a small difference between two largevalues, whereas fluorescence techniques measure an absolute value overan arbitrarily small background. Whether the detection apparatus is aquantitating optical device such as a spectrophotometer, or the humaneye, the signal to noise ratio is the limiting factor in determining thelevels of product detectable. For a reference that covers the basics offluorescence measurements see Guilbault, G., 1973, PracticalFluorescence: Theory, Methods and Techniques, Dekker, New York.

This extreme selectivity and sensitivity of fluorescence techniques maymanifest itself in several ways in GUS assays. First, the assays areremarkably fast. Because 100-1000 times less enzyme product than isrequired for a color production assay may be detected, results may beavailable 100-1000 times faster. Also, the quantity of material neededfor accurate and reliable assays is substantially reduced.

Whereas absorption methods produce a value which is absolute for a givencuvette cell size, and that can be used with a knowledge of theextinction coefficient of the compound to establish a concentration of asubstance according to the Beer-Lambert law, fluorescence output isproportional to excitation intensity and wavelength, and hence may notbe comparable between different machines, or even on different days forthe same machine without internal and reliable calibration of themachine with standard solutions. Calibration and expression of enzymeactivity in terms of absolute quantities of fluorochrome produced may bevery important for attaining reliable and reproducible results. It isoften true that fluorescence output is proportional to the concentrationof the fluorochrome, but this will depend on internal parameters, andmay only apply over a particular range of concentrations. In practicalterms however, these concentration ranges usually extend for at leastsix orders of magnitude.

Because fluorescence emission is dependent on the intensity andwavelength of the excitation light, any factors in an assay mixturewhich affect the available excitation intensity or wavelength willcorrespondingly affect the apparent fluorescence output. For instance,methylumbelliferone (MU--the product of MUG cleavage by GUS) absorbslight very effectively in the near ultraviolet range (UV) at 365 nm, andemits with a high quantum efficiency at 455 nm--in the blue range of thevisual spectrum. In concentration extracts made from plant leaves, thereis a significant level of chlorophyll, which has absorption maximaaround 400 nm, but with a broad spectral distribution; there appears tobe significant absorption at both 365 nm and at 455 nm. Becausechlorophyll absorbs some fraction of the excitation light, that light isnot available to excite the fluorochrome product of the GUS reaction,MU. Hence the intensity of the excitation light that is available toexcite the MU may be reduced, and the fluorescence of the MU in thesample lowered. In addition, the chlorophyll can absorb some of theemitted fluorescent light from the MU (at 455 nm), thereby reducingfurther the apparent fluorescence of the MU. This phenomenon is calledquenching. Quenching of fluorescence in extracts may be a problem if oneis unaware of it. However, it is a simple matter to control andeliminate quenching, either by reducing the concentration of extract,eliminating the absorbing and quenching molecules from the extract priorto assaying (e.g. by a spin-column) or by performing internalcalibrations with known quantities of MU in the same extract conditionsas the assay.

If background activity occurs, it maybe tracable to one of severalpotential sources. Often the background is intrinsic fluorescence thathas nothing to do with GUS, but simply shows the levels of endogenousfluorescent compounds. For example, occasionally callus, wounded plantcells (such as protoplasts that have been treated with polyethyleneglycol or electroporated) and tissues such as root, accumulatefluorogenic compounds (presumably secondary products, intermediates inlignin biosynthesis, and other phenylpropanoid pathway compounds). Someof these compounds are not fluorescent until after cell lysis which mayrelease enzymes that cleave off the glycoside or other conjugate torelease the free fluorochrome. This may be dealt with by the spin-columnmethod, or by following the kinetics of fluoroescence increase.

Background may also occur when using protoplasts to study transient geneexpression. Some enzyme mixtures used to prepare protoplasts have asizeable amount of a β-glucuronidase activity. The usual flotation andKCl washes work well to eliminate all residual activity from tobaccoprotoplasts, but other types of cells may be more or less difficult toclean up. There are also reports that some cell types, especially uponlengthy incubation with protoplasting enzymes, endocytose material fromthe medium. If this occurs, only the appropriate controls may minimizethe background, as washing may not help.

Another source of background may be the mutability of chemicals. Whenone performs extremely lengthy assays (overnight or longer) with veryconcentrated extracts, there is a likelihood that substrate may beconverted into another compound, and then cleaved or metabolized toproduce a fluorescent signal. This possibility may arise for anychemical reaction, but is best dealt with by using enzyme kinetics.

An additional potential problem is that bacterial GUS encoding genes maybe inducible by incubation with MUG. Hence, long assays of material thatis "contaminated" with bacteria may cause a bona fide GUS activity todevelop. Contamination of this kind may be unavoidable, even with veryclean tissues, as there may be endogenous bacterial and fungalpopulations in almost any plant. It may therefore be prudent toincorporate 0.02% NaN₃ in all lengthy assays to avoid this potentialproblem.

An additional and sometimes very useful technique is to use the specificβ-glucuronidase inhibitor saccharolactone (Levvy, G. A., 1952, Biochem.J. 52:464)(Sigma S-0375, saccharic acid 1-4 latone, glucaric acid 1-4lactone; glucarolactone) to corroborate the GUS-dependence of thefluorescence increase. This inhibitor will eliminate glucuronidaseactivity at concentrations less than one millimolar, but the compound isunstable at neutral pH, so that care should be exercised duringprolonged assays. Because of this instability, it is preferable to usesaccharolactone at up to 5 mM for assays up to half an hour.Alternatively, the reaction and the inhibited reaction may preferably beperformed at pH 6.0 or below. GUS activity should not be affected bythese conditions and saccharolactone is more stable at acid pH.

If the intrinsic fluorescence of the extract is a serious problemlimiting sensitivity, one may either extract the fluorescent compoundsprior to analysis (see above) or use another fluorogenic substrate.

Resorufin glucuronide, when hydrolyzed by GUS, yields resorufin, aphenoxazine derivative which has an extraordinarily high extinctioncoefficient and quantum efficiency, and excitation (570 nm) and emission(590) maxima conveniently in a range where plant tissue does not heavilyabsorb or fluoresce. These wavelengths are well separated by filter setsdesigned to optimize detection of the common fluorochrome rhodamine,making resorufin a useful fluorochrome for microscopic analysis, oranalysis using fluorimeters without monochromators. It fluorescesmaximally at neutral pH, with a pK_(a) of about 5.8, making itunnecessary to stop the reaction. Because of the tendency of resorufinto be reduced to a non-fluorescent form, it may be preferable to omitDTT or β-mercaptoethanol from the reaction mix. This tendency to beaffected by redox potential, and the price of the substrate are theprinciple disadvantages to the use of resorufin glucuronide.

4-Trifluoromethylumbelliferyl β-D-glucuronide is a very sensitivesubstrate for GUS. The fluorescence maximum is close to 500 nm--bluishgreen, where very few plant compounds fluoresce or absorb. TFMUG alsofluoresces much more strongly near neutral pH, allowing continuousassays to be performed more readily than with MUG. Importantly, TFMUGmay be used as a fluorescent indicator in vivo.

Substitution of the umbelliferone ring system in the 3-positiongenerally results in fluorochromes with a reduced pK_(a), hence betterfluorescence at neutral pH. In addition, many of these substitutions,such as 3-cyanoumbelliferyl β-D-glucuronide also have a higherextinction coefficient-in this case 3-4 times higher than MUG (Sherman,W. R. and Robins, E., 1986, Anal. Chem. 40:803-805).

Fluorescein is perhaps the most widely used fluorochrome in biology. Itfluoresces very well in living cells (Rotman, B. and Papermaster, B. W.,1966, Proc. Natl. Acad. Sci. U.S.A. 55:134-141), and is excited andemits at wavelengths that are spectrally neutral in plants. The mono anddi-glucuronides of fluorescein may be used as reagents for in vivo GUSanalysis.

5.9.6.2. Spectrophotometric Assay

The currently preferred substrate for spectrophotometric measurement isp-nitrophenyl β-D-glucuronide, which when cleaved by GUS releases thechromophore p-nitrophenol. At a pH greater than its pK_(a), (around7.15) the ionized chromophore absorbs light at 400-420 nm, giving ayellow color. Good color development can occur at the pH of the GUSreaction (7.0) but is enhanced and saturated by alkalinization of thereaction mixture. Perhaps the greatest advantage of using p-nitrophenolfor spectrophotometric assays is the ability to monitor the progress ofthe reaction continuously, and then to terminate the reaction byalkalinization when it has proceeded sufficiently.

Unfortunately, much plant tissue is rich with compounds that absorb atthe maximum wavelength of p-nitrophenol. Pigmented extracts can often beclarified and effectively decolorized prior to assay by passage througha spin-column of Sepharose CL6B. This treatment may be used to removelow or medium molecular weight chromophores that are not tightly boundto large macromolecules. Alternatively, other chromogenic substrates maybe used.

The chromogenic substrate phenophthalein β-D-glucuronide is widely used;phenolphthalein is a deep red chromophore under alkaline conditions.This substrate is not in wide use now, due in part to its expense and tohe very low relative V_(max) for E. coli GUS (about 30 times lower thanthat for p-nitrophenyl-β-D-glucuronide) (Tomasic and Keglevic, 1973,supra), but it can still be a useful compound under conditions where theyellow of p-nitrophenol is inappropriate or difficult to detect.Phenolphthalein β-glucuronide does not induce the GUS operon of E. coli,whereas p-nitrophenyl-β-glucuronide induces the operon very well. Henceuse of p-nitrophenyl β-glucuronide in bacterial-cultures will bothinduce GUS and permease, and assay their function in vivo. This shouldbe remembered when performing assays using p-nitrophenyl glucuronide (ormethylumbelliferyl glucuronide or 5-bromo-4-chloro-3-indolylglucuronide) on extracts or tissues that may have E. coli or other GUS⁺bacteria as contaminants.

The spectrophotometric assay is very straightforward and moderatelysensitive (Jefferson et al., 1986, Proc. Natl. Acad. Sci. U.S.A.86:8447-8451). Because of the remarkable stability of GUS, one canenhance the sensitivity quite significantly by using very long assays(overnight assays may preferably be used to provide linear, reproducibleresults). The assay is also inexpensive, easy to automate and easy toquantitate without sophisticated instrumentation. Its limitations arethe intrinsic lack of sensitivity of methods based on absorption oflight (which measure a small difference in two large numbers), and theproblems caused by light absorption by pigments in extracts. This assaymay be automated using commercially available ELISA plate readers andmicrotiter equipment.

For very long assays (a few hours or longer), it is preferable to add0.02% NaN₃ to prevent microbial growth or induction of microbial GUS,and 100-200 μg.ml BSA (bovine serum albumin) to stabilize the enzyme andwith endogenous proteases and oxidizing agents.

5.9.7. Histochemical Assays of GUS

Detailed description of laboratory methods for histological andmicroscopic manipulation of plants, including preparation of materials,sectioning tissues by hand, and microscopic analysis is given byO'Brien, T. P. and McCully, M. E., 1981, The Study of Plant Structure:Principles and Selected Methods. Termarcarphi Pty. Ltd., Melbourne,Australia. Microscopic analysis of plant tissues generally involves thepreparation of thin sections of material that can transmit a certaindegree of light, and hence information about the specimen. When a liveorganism is killed and tissue sections are generated for microscopicanalysis, many changes occur within the specimen that do not generallyreflect the state of the living system. Cell contents leak out and mixwith each other, ultrastructure is altered, degradative enzymes begin todestroy the macromolecular components, and chemical changes occur.Fixation is the compromise that must be reached to minimize thisperturbation. If it were possible to maintain the cellular contentswithin the cell, that is, fix them, but not lose their biologicalactivity, and if the cellular and subcellular architecture of the livingspecimen could be effectively maintained, we would be able to examinethe biological activity in situ, and make useful inferences about thestructure and function of the live organism. However, in treating atissue or tissue section with a chemical compound designed to cross-linkor coagulate proteins and other macromolecules, there is clearly a largepotential for distortion of the natural specimen. Whereas aheavily-fixed specimen may retain excellent morphology, it may havelittle, if any remaining biological or enzymatic activity. Therefore,achieving a suitable balance between the preservation of morphology andthe preservation of enzyme activity should be the ultimate aim of afixation protocol. Clearly this will have to be determined empirically.However, a sound understanding of both the chemistry of fixation, andthe properties of the specimen and enzyme (i.e., GUS) is invaluable.

Fixation conditions will vary with the fixative, the sample, the tissue,the cell-type, its permeability to the fixative and other variables ofeach experiment.

Glutaraldehyde effectively cross-links proteins and othermacromolecules, but does not easily penetrate leaf cuticle although itis immediately taken up through cross-sections. GUS seems to berelatively sensitive to inactivation by gluaraldehyde treatment.Formaldehyde is a more gentle fixative than glutaraldehyde forpreservation of GUS activity; it penetrates most plant tissues verywell, and can be used for longer periods. Although glutaraldehyde isvery effective at preserving structures in specimens, it is, for thesame reasons, a very potent agent for inactivating enzymes, includingGUS.

For treatment of protoplasts, which present very little permeabilitybarrier to fixatives, 1% formaldehyde in 0.3-0.6M mannitol, 10 mM MES,pH 5.6 for 30-60 minutes at 0°-4° C. may be used. This should befollowed by several washes for 10-15 minutes each, usually in phosphatebuffer and/or osmoticum, to remove fixative. Almost all starting GUSactivity should be maintained under these conditions, although this maybe measured with suitable controls for each new situation.

For fixation of other plant tissues, 1-2% formaldehyde (prepared bydilution of reagent grade 37T formalin solution) in a 50 mM NaHPO₄buffer at ph 7.0, containing 0.05% Triton X-100 may be used. The TritonX-100 serves to help wet the surface of the specimen, aiding in uptakeof the fixative.

The best substrate currently available for histochemical localization ofβ-glucuronidase activity in tissues and cells is5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) (Holt, S. J. andWithers, R. F. J., 1958, Proc. Roy. Soc. B. 148:520-534; Anderson, F. B.and LeEback, D. H., 1961, Tetrahedron, 12:236-239; Pearson, et al.,1963, Lab. Invest. 12:1249-1259; Pearson, et al., 1967, Lab. Invest.17:217-224; Yoshida et al., 1975, Chem. Pharm. Bull. 23:1759; Jeffersonet al., 1986, Proc. Natl. Acad. Sci. U.S.A. 86:8447-8451). Thissubstrate gives a blue precipitate at the site of GUS enzyme activity.There are numerous variables that affect the quality of thehistochemical localization, including all aspects of tissue preparationand fixation as well as the reaction itself.

It is worthwhile understanding the nature of the reaction to bettercontrol the variables. The product of glucuronidase action on X-Gluc isnot colored. Instead, the indoyl derivative produced must undergo anoxidative dimerization to form the very insoluble and highly coloredindigo dye, 5,5'-dibromo 4,4'-dichloro indigo. This dimerization isstimulated by atmospheric oxygen, and may be enhanced by using anoxidation catalyst such as a K⁺ ferricyanide/ferrocyanide mixture (Holtand Withers, 1958, supra; Lojda, 1970, Histochemie 23:266-288; reviewedin Pearse, 1976). Without such a catalyst, the localized peroxidases mayenhance the apparent localization of glucuronidase. One should not getfalse positives, but the relative degree of staining may not necessarilyreflect the concentrations of glucuronidase.

An alternative histochemical assay for GUS uses naphtholASBI-glucuronide, cleaved to liberate the free naphthol ASBI, thencoupled to a diazo dye.

Incubate the formaldehyde or glutaraldehyde fixed tissue or whole mountsin 0.1M NAPO4 pH 7.0 with 1 mM Napthol ASBI glucuronide in a moistchamber at 37° C. For very low amounts of enzyme lengthy incubation maybe necessary, but may give poorer localization of activity due todiffusion of the primary reaction product. The specimen may then bewashed in phosphate buffer and coupled using a fresh solution ofdiazotized dye in phosphate buffer. Post-coupling with a 1-5 mg/mlsolution of Fast Garnet GBC in phosphate buffer, pH 7, produces a resultafter as little as thirty seconds coupling, but many different couplingagents may be used. There is a voluminous literature about thesehistochemical methods as applied to mammalian glucuronidases (reviewedin Pearse, 1976, supra).

The GUS substrates and assays described herein are only a few of theexamples of substrates and assays useful in conjunction with the GUSgene fusion system, and are not limiting of the scope of the invention.

6. EXAMPLE: CLONING OF THE ESCHERICHIA COLI GENE FOR BETA-GLUCURONIDASE6.1. Materials and Methods

6.1.1. DNA Manipulation

Restriction enzymes and DNA Modifying enzymes were obtained from NewEngland Biolabs whenever possible and used as per the instructions ofthe manufacturer. Plasmid DNA preparations were done by the method ofBirnboim and Doly (Birnboim, et al., 1979, Mucleic Acids Res. 7:1513) asdescribed in Maniatis et al. (Maniatis, et al. 1982, Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.)). Routine cloning procedures, including ligations andtransformation of E. coli cells, were performed essentially as describedin Maniatis, et al. 1982, Molecular Cloning: A Laboratory Manual (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.). DNA fragments werepurified from agarose gels by electrophoresis onto Schleicher & SchuellNA 45 DEAE membrane (Dretzen, et al., 1981, Anal. Biochem. 112:295-298)as recommended by the manufacturer. DNA sequences were determined by thedideoxy chain terminator method of Sanger and Coulson (Sanger et al.,1975, J. Mol. Biol. 95:441) as modified by Biggin et al. (Biggin, etal., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:3963-3965). Oligonucleotideprimers for sequencing and site-directed mutagenesis were synthesizedusing an Applied Biosystems DNA synthesizer, and purified by preparativepolyacrylamide gel electrophoresis. Site directed mutagenesis wasperformed on sDNA obtained from pEMBI derived plasmids, essentially asdescribed in Zollen et al. (1982), Nucl. Acids Res. 10:6487-6500). Thestrain used for routine manipulation of the uidA gene was RJ21, a recAderivative of JM83 (Viera, et al., 1982, Gene 19:259-268) generated byP1 transduction. Strain PK803 was obtained from P. Keumpel of theUniversity of Colorado at Boulder, and contains a deletion of themanA-uidA region. Plasmid vectors pUC7, 8 and 9 (Herrera-Estrella, etal., 1983, EMBO J. 2:987-995) and pEMBL (Dente, et al., 1983, NucleicAcids Res. 11:1645) have been described.

6.1.2. Protein Sequencing and Amino Acid Analysis

Sequence analysis was performed by Dr. A. Smith of the Protein StructureLaboratory, University of California, Davis, using a Beckman 890Mspinning-cup sequenator. Amino acid composition was determined byanalysis of acid hydrolyzates and purified beta-glucuronidase on aBeckman 6300 amino acid analyzer.

6.1.3. Protein Analysis

Protein concentrations were determined by the dye-binding method ofBradford (Bradford, 1976, Anal. Biochem. 72:248) using a kit supplied byBIO-RAD Laboratories. Sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) was performed using the Laemmli system(Laemmli, 1970, Nature 227:680).

6.1.4. Beta-Glucuronidase Assays

Glucuronidase was assayed in a buffer consisting of 50 mM NAPO₄, pH 7.0,10 mM R-ME, 0.1% Triton X-10, 1 mM p-nitrophenyl beta-D-glucuronide.Reactions were performed in one ml volumes at 37° C., and terminated bythe addition of 0.4 ml 2.5M 2-amino-2-methyl propanediol. p-Nitrophenolabsorbance was measured at 415 nm. Routine testing of bacterial coloniesfor beta-glucuronidase activity was done by transferring bacteria with atoothpick into microtiter wells containing the assay buffer. Thehistochemical substrate 5-bromo, 4-chloro, 3-indolyl beta-D-glucuronide(analogous to the beta-galactosidase substrate X-gal) is commerciallyavailable (Research Organics Inc., Cleveland, Ohio), and is found to bean excellent and sensitive indicator of beta-glucuronidase activity insitu when included in agar plates at a concentration of 50 μg/ml.

6.1.5. Purification of Beta-Glucuronidase

Beta-glucuronidase was purified by conventional methods from the strainRJ21 containing the plasmid pRAJ210. FIG. 1 illustrates subcloning andstrategy for determining the nucleotide sequence of the uidA gene.Briefly, pBKuidA was generated by cloning into pBR325. pRAJ210 andpRAJ220 were generated in pUC9, with the orientation of the uidA geneopposite to that of the lacZ gene in the vector. The sequence wasdetermined from both strands for all of the region indicated except fromnucleotide 1 or 125. The orientation of the coding region is from leftto right.

6.2. Results

6.2.1. Subcloning and Sequencing of the uidA Gene

The starting point for the subcloning and sequencing of thebeta-glucuronidase gene was the plasmid pBKuidA shown in FIG. 1. pBKuidAhas been shown to complement a deletion of the uidA-manA region of theE. coli K-12 chromosome restoring beta-glucuronidase activity whentransformed into the deleted strain, PK803. The strategy for thelocalization of the gene on the insert is shown in FIG. 1.

A restriction map of the insert was obtained, and various subclones weregenerated in the plasmid vector pUC9, and tested for their ability toconfer beta-glucuronidase activity upon transformation of PK803. Theintermediate plasmid pRAJ210 conferred high levels of glucuronidaseactivity on the deleted strain, and was used for the purification of theenzyme. Several overlapping subclones contained within an 800 base pair(bp) Eco RI-Bam HI fragment conferred high levels of constitutivebeta-glucuronidase production only when transformed into a uidA⁺ hoststrain, and showed no effect when transformed into PK803.

It is surmised that the 800 bp fragment carried the operator region ofthe uidA locus, and was possibly titrating repressor to give aconstitutively expressing chromosomal uidA⁺ gene. With this informationto indicate a probable direction of transcription, and a minimum genesize estimate obtained from characterization of the purified enzyme, aseries of BAL 31 deletions were generated from the Xho I site ofpRAJ210. The fragments were gel purified, ligated into pUC9 andtransformed into PK803. The resulting colonies were then assayed forbeta-glucuronidase activity. .

The smallest clone obtained that still gave constitutive levels ofbeta-glucuronidase was pRAJ220, which contained a 2.4 kilobase pair (kb)insert. Subclones of this 2.4 kb fragment were generated in M13mp8 andmp9 and their DNA sequence was determined as illustrated in FIG. 2.

FIG. 2 shows the DNA sequence of the 2439 bp insert of pRAJ220,co-tailing the beta-glucuronidase gene. The arrows before the codingsequence indicate regions of dyad symmetry that could be recognitionsequences for effector molecules. The overlined region is the putativeShine/Dalgarno sequence for the uidA gene, while the brackets indicatetwo possible Pribnow boxes. All of the palindromic regions fall withinthe smallest subcloned region (from the Sau 3A site at 166 to the Hind Isite at 291) that gave constitutive genomic expression of uidA whenpresent in high copy in trans, consistent with their proposed functionas repressor binding sites. The terminator codon at 2106 overlaps withan ATG that may be the initiator codon of a second open reading frame,as indicated (see Discussion).

6.2.2. Manipulation of the uidA Gene for Vector Construction

The plasmid pRAJ220 contains the promoter and operator of the E. coliuidA locus, as well as additional out-of-frame ATG codons that wouldreduce the efficiency of proper translational initiation of eukaryoticsystems (Kozak, 1983, Microbiol. Rev. 47: 1-45). It was necessary toremove this DNA to facilitate using the structural gene as a reportermodule in gene fusion experiments. This was done by cloning andmanipulating the 5' region of the gene separately from the 3' region,then rejoining the two parts as a lacZ:uidA fusion that showedbeta-glucuronidase activity under lac control. The resulting plasmid wasfurther modified by progressive subcloning, linker additions andsite-directed mutagenesis to generate a set of useful gene modulevectors. These manipulations are illustrated by reference to FIG. 3,which shows GUS gene module vectors.

As illustrated in FIG. 3, pRAJ220 (see FIG. 1) was digested with Hind I,which cleaves between the Shine/Dalgarno sequence and the initiator ATG,the single-stranded tails were filled in, digested with Bam HI and theresulting 515 bp fragment was gel purified and cloned into pUC9/Hind IIand Bam HI. This plasmid was digested with Bam HI and the 3' region ofthe uidA gene carried on a 1.6 kb Bam HI fragment from pRAJ220 wasligated into it. The resulting plasmid, pRAJ230, showed IPTG inducibleGUS activity when transformed into JM103. pRAJ230 was further modifiedby the addition of Sal 1 linkers to generate pRAJ240, an in-framelacZ:uidA fusion in pUC7. pRAJ230 was digested with Aat II, which cuts45 bp 3' to the uidA translational terminator, the ends were filled,digested with Pst I, and the resulting 1860 bp fragment was gelpurified, and cloned into pEMBL9/Pst I and Sma I. The resulting plasmid,pRAJ250, is an in-frame lacZ:uidA fusion. The Bam HI site that occurswithin the coding region at nucleotide 807 was eliminated byoligonucleotide-directed mutagenesis of single-stranded DNA preparedfrom pRAJ250, changing the Bam HI site from GGATCC to GAATCC, with nochange in the predicted amino acid sequence. The clone resulting fromthe mutagenesis, pRAJ255, shows normal GUS activity, and lacks the BamHI site. This plasmid was further modified by the addition of ^(a) Pst Ilinker to the 3' end and cloned into pEMBL9/Pst I, to generate pRAJ260.

6.2.3. Purification and Properties of Beta-Glucuronidase

Beta-glucuronidase activity in E. coli is induced by a variety ofbeta-glucuronides; methyl glucuronide is among the most effective(Stoeber, 1961, These de Docteur es Sciences, Paris). To determine thesize and properties of the enzyme and to verify that the enzyme producedby the clone pRAJ210 was in fact the product of the uidA locus, theprotein was purified from the over-producing strain, and the purifiedproduct was compared with the enzyme induced from the single genomiclocus by methyl glucuronide.

Aliquots of supernatants from induced and uninduced cultures of E. coliC600 were analyzed by SDS-PAGE and compared with aliquots of thepurified beta-glucuronidase as shown in FIG. 4. In FIG. 4 lane (a) ismolecular weight standards; lane (b) is extract from uninduced C600;lane (c) is extract from C600 induced for beta-glucuronidase with MeGlcU(see reference 23); lane (d) is 0.3 μg of purified beta-glucuronidase(calculated to contain the same activity as the induced extract); lane(e) is 3.0 μg aliquot of purified beta-glucuronidase.

The induced culture of C600 shows only a single band difference relativeto the uninduced culture. The new band co-migrates with the purifiedbeta-glucuronidase, indicating that the enzyme purified from the overproducing plasmid has the same subunit molecular weight as the wild-typeenzyme.

The purified enzyme was analyzed for amino acid composition andsubjected to eleven cycles of Edman degradation to determine the aminoterminal sequence. The amino acid composition agrees with the predictedcomposition derived from the DNA sequence, and the determined amino acidsequence agrees with the predicted sequence, identifying the site oftranslational initiation and indicating that the mature enzyme is notprocessed at the amino terminus.

E. coli beta-glucuronidase is a very stable enzyme, with a broad pHoptimum (from pH 5.0 to 7.5); it is half as active at pH 4.3 and pH 8.5as at its neutral optimum, and it is resistant to thermal inactivationat 50° C.

6.3. Discussion

6.3.1. Molecular Analysis of the uid Locus

The complete nucleotide sequence of the E. coli uidA gene, encodingbeta-glucuronidase, has been determined. The coding region of the geneis 1809 bp long, giving a predicted subunit molecular weight for theenzyme of 68,200 daltons, in agreement with the experimentallydetermined value of about 73,000 daltons. The translational initiationsite was verified by direct amino acid sequence analysis of the purifiedenzyme.

Genetic analysis of the uidA locus has shown three distinct controllingmechanisms, two repressors and a cAMP dependent factor, presumably CAP(Novel, et al., 1976, J. Bacteriol. 127:418-432). The DNA sequencedetermined includes three striking regions of dyad symmetry that couldbe the binding sites for the two repressors and the CAP protein. One ofthe sequences matches well with the consensus sequence for CAP binding,and is located at the same distance from the putative transcriptionalinitiation point as the CAP binding site of the lac promoter. It isinteresting that the putative CAP Binding site overlaps one of the otherpalindromic sequences, suggesting a possible antagonistic effect of CAPand one or both repressors.

The sequence analysis indicates the presence of a second open readingframe of at least 340 bp, whose initiator codon overlaps thetranslational terminator of the uidA gene. This open reading frame istranslationally active. Although a specific glucuronide permease hasbeen described biochemically (Stoeber, 1961, These de Docteur esSciences, Paris), the level of genetic analysis performed on the uidlocus would not have distinguished a mutation that eliminatedglucuronidase function from a mutation that eliminated transport of thesubstrate (Novel, et al., 1973, Mol. Gen. Genet. 120:319-335; Novel, etal., 1974, J. Baceriol. 120:89-95). All mutations that specificallyeliminated the ability to grow on a glucuronide mapped to the uidAregion of the E. coli map, indicating that if there is a generesponsible for the transport of glucuronides, it is tightly linked touidA. By analogy to the lac operon, it is proposed that the coupled openreading frame may encode a permease that facilitates the uptake ofbeta-glucuronides.

6.3.2. THE uidA Gene as Gene Fusion Marker

Plasmid vectors have been constructed in which the uidA structural genehas been separated from its promoter/operator and Shine/Dalgarno region,and placed within a variety of convenient restriction sites. The GUSgene on these restriction fragments contains all of thebeta-glucuronidase coding information, including the initiator codon;there are no ATGs upstream of the initiator. These vectors allow theroutine transfer of the beta-glucuronidase structural gene to thecontrol of heterologous sequences, thereby facilitating the study ofchimeric gene expression in other systems.

The uidA encoded beta-glucuronidase is functional with severalcombinations of up to 20 amino acids derived from the lacZ gene and/orpolylinker sequences. Translational fusions to GUS have also been usedsuccessfully in transformation experiments in the nematodeCaenorhabditis elegans, and in Nicotiana tabacum, giving enzyme activitywith many different combinations of amino terminal structures (seebelow).

7. EXAMPLE: EXPRESSION OF BETA-GLUCURONIDASE GENE FUSIONS INCAENORABDITIS ELEGANS

Experiments have also been performed which show the expression oftransformed genes in the nematode Caenorhabditis elegans using the genefusion system. In particular, gene fusions between GUS and twowell-characterized genes of C. elegans, col-1 and MSP (p3L4) have beenconstructed. col-1 is a collagen gene that is transcribed predominantlyin embryos, and somewhat less in later developmental stages, andpresumably encodes a component of the first larval stage cuticle.

The MSP gene P3L4 is a transcribed member of the major sperm proteingene family, which encodes a set of abundant, closely related 15,000Mproteins that are present only in sperm. The MSP genes are transcribedonly during spermatogenesis, which occurs during the fourth larval stage(L4) in hermaphrodites and in L4 and adult stages in males. Briefly,vectors consisting of the flanking regions of the collagen gene (col-1)or major sperm protein gene of C. elegans fused to the Escherichia coliuidA gene, encoding beta-glucuronidase, were microinjected into wormsand found to be propagated as high-copy extrachromosomal tandem arrays.Beta-glucuronidase activity was detected in transformed lines, and theactivity has been shown to be dependent upon the correct reading frameof the construction and on the presence of the worm sequences. Theenzyme activity was shown to be encoded by the chimericbeta-glucuronidase gene by co-segregation analysis and by inactivationwith specific antisera. Expression is at a very low level, and seems tobe constitutive. Histochemical techniques have been used to visualizethe enzyme activity in embryos.

7.1. Materials and Methods

7.1.1. DNA Constructs

The structures of the in-frame col-1:GUS fusion pRAJ321 and the in-frameMSP:GUS fusion pRAJ421 are shown in FIG. 5. In FIG. 5 both vectors arebuilt within pUC9 (Vieira and Messing, 1982, Gene 19:259-268). Thehatched region is the lac-derived sequence from pUC9. pRAJ321 is 5.8 kband pRAJ421 is 5.5 kb. DNA manipulations were performed essentially asdescribed in Maniatis et al. supra. pRAJ321 encodes the first 5 aminoacids of the col-1 gene product plus 9 amino acids derived from linkersequences fused in-frame to the entire coding region of the E. coli uidAgene, and followed by the 3' intron, the translational termination codonand the polyadenylation signal from the col-1 gene. The col-1 promotermodule extends from the Hinc II site 530 bp upstream from thetranscription initiation site to a Bal31 generated breakpoint 14 bp intothe protein coding sequence of col-1. This fragment cloned into the HindIII and Pst I sites of pUC9 is designated pRAJ301. pRAJ303 was generatedby elimination of the promoter-proximal Pst site of pRAJ301, andinsertion of an octameric Pst I linker. The 3' intron and thepolyadenylation site from the col-1 gene are contained on a 436 bp PvuII-Hind III fragment cloned into the Sma I site of pUC9, designatedpRAJ310. The 570 bp Hind III-Sal I fragment from pRAJ301 and pRAJ303were cloned into pRAJ310 to generate the expression vectors, pRAJ311,and pRAJ313. The MSP:GUS fusion vector pRAJ421, encodes the initiatormethionine of the MSP coding sequence, 9 amino acids derived from linkersequences, and the GUS coding region, followed by the translationalterminator of the MSP gene and the polyadenylation signal. The promotermodule extends from a Hind III site 584 bp upstream from the initiatorATG to a Hind III site 4 bp into the coding sequence. Using a Pst Ilinker, this fragment was cloned into the Hind III-Pst I sites of pUC9and is designated pRAJ401. The MSP terminator module extends from theRsaI site located 8 nucleotides upstream from the translation terminatorcodon, to the Hind III site, a total of about 150 bp. This fragment wasshown to contain the polyadenylation site. After several manipulations,the resulting 160 bp fragment was cloned into pRAJ401 that had beendigested with Bam HI and Sma I. A plasmid clone was obtained thatcontained the terminator in the correct orientation to the promoter,designated pRAJ411. The uidA structural gene encoding beta-glucuronidase(GUS) was transferred into the expression vectors as a 2.1 kb Sal Ifragment from the plasmid pRAJ240. Clones in the correct orientationwere obtained, and the nucleotide sequences at the junctions weredetermined using specific oligonucleotide primers complementary to the5' coding region of the uidA gene. pRAJ421 and pRAJ321 were shown tohave GUS in-frame to the MSP and col-1 initiators, respectively, whileGUS in pRAJ323 was shown to be out-of-frame with respect to the col-1initiator.

7.1.2. Transformation with Plasmid DNA

Plasmid DNA was injected into the distal gonal arm of adulthermaphrodite worms at a concentration of approximately 500 μg/ml,essentially as described in Stinchomb et al., 1985, Mol. Cell. Biol.5:3484-3496. The strain used was DH408, lacking glucuronidase activity(Horch et al., 1984, Science 223:496-498). Lines carrying the injectedDNA as high-copy extrachromosomal tandem arrays were obtained from theF2 generation of the injected worms. Stability and physical propertiesof the tandem arrays were similar to those described in Stinchcomb etal., supra. Transformants were obtained containing either an in-framecol-l:GUS fusion (pRAJ321), an out-of-frame col-1:GUS fusion (pRAJ323),an in-frame MSP: GUS fusion (pRAJ421) or a GUS-encoding plasmidcontaining no worm sequences (pRAJ210).

7.1.3. Fluorometric Assays

Fluorometric assays were performed in 100 μl of 50 mM-NaPO₄ (pH 7.0), 10mM beta-mercaptoethanol, 0.1% (v/v) Triton X-100, 0.5 mM-4-methylumbelliferyl beta-D-glucuronide, at 37° C., and terminated with theaddition of 1 ml of 0.2M-Na₂ CO₃, or 100 μl of 1M-Na₂ CO₃ (forsmall-scale, qualitative assays). The reaction products were visualizedby irradiation with 365 nm light (for qualitative assays) or byfluorescence measurements with ex³⁶⁵ nm and em⁴⁵⁵ nm. Extracts wereprepared from worms harvested from Petri plates, washed twice in M9salts, resuspended in assay buffer without substrate and passed througha French pressure cell at 12,000 lb/in². Protein concentrations wereadjusted to 20 mg/ml and 50 μl portions were assayed. Measurements weremade on a Perkin-Elmer LS-3 spectrofluorometer. Worms from thepopulations harvested were shown to contain the transforming DNA atsimilar copy number. Extracts prepared as described above were incubatedwith equal volumes of either neat pre-immune serum, or affinity-purifiedantibody directed against purified E. coli beta-glucuronidase, at aconcentration of 125 μg/ml for 3 hours at 4° C. Protein A-Sepharose wasadded, and the reactions were allowed to sit for 2 hours at 4° C. Theextracts were centrifuged at 12,000 g for 5 minutes, and then assayedfor beta-glucuronidase. Worms were grown for enzyme assays on the E.coli strain PK803 (obtained from P Kuempel) that contains a deletion ofthe uidA locus and has no detectable glucuronidase activity.

7.2. Results and Discussion

Extracts were prepared from populations of transformed worms and assayedfor the presence of beta-glucuronidase. The results are shown in FIG. 6.

Extracts from uninjected worms, worms carrying the out-of-framecol-1:GUS fusion, or pRAJ210 showed no detectable beta-glucuronidaseactivity, while extracts from worms carrying either the in-framecol-1:GUS fusion or the MSP:GUS fusion showed significant levels ofenzyme activity. Reconstruction experiments with purifiedbeta-glucuronidase in worm extracts indicated that the quantities ofenzyme in the transformed extracts were about 1 to 2 ngbeta-glucuronidase/mg soluble protein, assuming comparable turnovernumbers of the native and chimeric enzymes. Consistent with theextremely low enzyme levels, no transcript was detected by Northern blotanalysis, nor has an immunologically cross-reactive protein beendetected in extracts, as judged by Western blots, to a sensitivity ofabout one part in 10⁵.

To verify that the beta-glucuronidase activity measured in the extractswas due to the E. coli uidA gene product, portions of the extracts wereincubated with antibody to homogeneous E. coli beta-glucuronidase orwith pre-immune serum. Immune complexes were precipitated with StaphA-Sepharose, and the supernatant was assayed for beta-glucuronidaseactivity (FIG. 6). Beta-glucuronidase activity diminished only afteraddition of antibody directed against purified bacterial glucuronidase;preimmune serum showed no effect.

It was possible that a small number of integrated copies of the chimericgenes was responsible for the observed enzyme activity, and that thelarge tandem array was inactive. To test this possibility, 40 F2 wormsfrom a transformed individual were cloned and grown to saturation onplates. Extracts were prepared and assayed for the presence of thetransforming DNA by dot blot hybridization, and for beta-glucuronidaseactivity.

The 40 F2 worms derived from a transformant carrying the col-1:GUSfusion were cloned onto individual Petri plates, grown to saturation,harvested and washed, and the culture was split into 2 parts. Extractswere prepared for (a) fluorogenic beta-glucuronidase assays or (b) DNAdot bots. Glucuronidase assays were performed essentially as describedin Connection with FIG. 6, in the wells of a Gilson tube rack at 37° C.for 4 hours, and visualized by addition of Na₂ CO₃ and placing the rackon a long-wavelength ultraviolet light box. DNA dot blots were probedwith ³² P-labelled pRAJ210. All worm cultures that gave rise to apositive signal by DNA dot blot analysis also gave rise to glucuronidaseactivity, and vice versa. In several repeats of this experiment no casewas observed in which strict co-segregation of the high-copy DNA andbeta-glucuronidase activity was not maintained. The results are shown inFIG. 7.

The high-copy transforming DNA and the enzyme activity alwaysco-segregated, indicating that the extra chromosomal tandem array wasresponsible for the beta-glucuronidase activity. Identical results wereobtained from transformed populations carrying either the MSP:GUS or thecol-1:GUS fusions.

To determine whether temporal regulation of the transforming DNA wasoccurring, extracts from staged populations of transformed worms wereassayed for beta-glucuronidase activity. For both the col-1:GUS fusionand the MSP:GUS fusion, specific activity of β-glucuronidase was highestin embryos, and decreased with developmental time. The temporal patternof expression of the col-1:GUS fusion is consistent with the availabledata on col-1 expression as determined by DNA dot blots in Northernblots (Kramer et al., 1985, J. Biol. Chem. 260:1945-1951). However, thepattern is also consistent with a low level constitutive expression ofthe chimeric gene when accounting for the near 100-fold increase in theprotein content of the worm during development. In the case of the majorsperm protein gene fusion, this temporal pattern of expression isinconsistent with the normal expression of MSP genes only during the L4stage, but is consistent with constitutive expression of the chimericgene.

In order to visualize beta-glucuronidase activity in situ, embryos wereprepared from a population of worms containing the in-frame col-1:GUSfusion and an untransformed control population, fixed and assayedhistochemically for beta-glucuronidase activity.

Freeze-cracked, formaldehyde-fixed (3% (w/v) paraformaldehyde inphosphate buffer (pH 7) for 3 minutes on ice) embryos from DH408 (a) orDH408 containing pRAJ321 (b) were assayed for glucuronidase activityusing naphthol-ASB1 glucuronide for 6 hours at 37° C., and post-coupledwith freshly prepared hexazonium pararosanalin (Fishman et al., 1965, J.Histochem. Cytochem. 13:441-447). The results are shown in FIG. 8.

In the transformed population (a), many embryos show the red precipitatecharacteristic of beta-glucuronidase activity, while the untransformedpopulation (b) never shows staining. The number of positives in a giventransformed population, and the intensity of staining within apopulation, varies considerably. Larvae and adults from a transformedpopulation assayed under similar conditions did not show detectablestaining. Perhaps because the low levels of beta-glucuronidase in thetransformed populations it has not been possible to localize theactivity spatially within the embryo, either histochemically or byindirect immunofluorescence or immunocytochemistry. Under the fixationconditions and lengthy assay times used to obtain histochemical stainingof the transformed embryos (due to the low levels ofbeta-glucuronidase), the diffusion of the product may be preventingdiscrete localization, if indeed it is occurring.

In summary, the GUS fusion system has been used to measure chimericenzyme levels in transformed worms. The expression of GUS in thetransformed lines is dependent upon the presence of worm promoters, andon the correct reading frame of the translational fusions used. Thelevels of expression in these transformants are very low, but easilymeasured using a fluorometric enzyme assay for beta-glucuronidase,corresponding to about one part in a million of the soluble protein in aworm extract. The transforming DNA is in the form of long extrachromosomal tandem arrays, a situation that certainly does not mimic thenormal in vivo condition of the genes under study. Possibly thestructure of the arrays imposes characteristic restrains on theexpression of genes within them, perhaps due to chromatin structure or apeculiarity of conformation. It is possible that integration of thetransforming DNA will allow higher levels of expression. Methods haverecently been developed to allow integration of exogenous DNA in C.elegans. Integration of the vectors described here into the germline ofthe worm may allow resolution of whether the low level, andinappropriate developmental expression of chimeric genes, is due totheir extra chromosomal tandem-array structure or to some other featureof the constructions.

8. EXAMPLE: EXPRESSION OF BETA-GLUCURONIDASE GENE FUSIONS IN HIGHERPLANTS 8.1. Materials and Methods

8.1.1. Nucleic Acid Manipulation

DNA manipulations were performed essentially as described in Maniatis etal., (1982, in "Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.). Enzymes were obtained fromNew England Biolabs, Boehringer-Mannheim or BRL.

8.1.2. Plant Transformation and Regeneration

Binary vectors containing CaMV-GUS fusions and rbcS-GUS fusions in E.coli MC1022 were mobilized into Agrobacterium tumefaciens LBA4404 asdescribed in Bevan, 1984, Nucl. Acids Res. 12: 8711-8721. The integrityof the vector in Agrobacterium was verified by preparing DNA fromAgrobacterium immediately before plant transformation using the boilingmethod (Mones and Quigley, 1981, Anal. Biochem. 114:193-201). Leaf discsof Nicotiana tabacum, var. Samsun were transformed using the leaf discmethod (Horsch et al., 1984, Science 223: 496-498) , and transformedplants were selected on MS medium (Marashige and Skoos, 1962, Physiol.Plant .15: 473 ) containing 100 μg/ml kanamycin. Plants were maintainedin axenic culture on MS basal medium, 3% sucrose, 200 μg/mlcarbenicillin and 100 μg/ml kanamycin, at approximately 2000 lux, 18hour day, 26° C.

8.1.3. Southern Blot Analysis

DNA was prepared from plants by phenol extraction and ethanolprecipitation of plant homogenates, followed by RNAse digestion, phenolextraction and isopropanol precipitation. Extracts were prepared fromaxenic tobacco plants using approximately 100 mg fresh weight of tissueground in 500 μl extraction buffer. Ten μl of extract was incubated at37° C. in 4 ml assay buffer and 1.0 ml aliquots were withdrawn at 0, 5,10 and 15 minutes intervals and stopped by addition to 1 ml 0.2M Na₂CO₃. The fluorescence of liberated 4-MU was determined as described. Oldleaves were lower, full-expanded leaves approximately 5 cm long, whileyoung leaves were approximately 5 mm long, and were dissected from theshoot apex. All samples were taken from the same plant (either CaMV-GUS21, SSU GUS 2 or non-transformed) at the same time. DNA samples (10 μgwere digested with restriction endonucleases, electrophoresed in an 0.8%agarose gel and blotted onto nitrocellulose (Maniatis et al., supra).Filters were hybridized with oligomer-primed, ³² P labelled GUS genefragment (Feinberg and Vogelstein, 1984, Anal. Biochem. 137:266-269) andthen washed with 0.2% SSC at 65° C.

8.1.4. Substrates

Substrates included: 4-methyl umbelliferyl glucuronide (MUG) (SigmaM-9130), 5-bromo-4-chloro-3-indolyl beta-D-glucuronide (X-GLUC)(Research Organics Inc., 4353 E. 49th St., Cleveland, Ohio, U.S.A.),resorufin glucuronide (ReG) (Molecular Probes Inc., 4849 Pitchford Ave.,Eugene, Oreg., U.S.A.).

8.1.5. Lysis Conditions

Tissues were lysed for assays into 50 mM NaH₂ PO₄ pH 7.0. 10 mM EDTA,0.1% Triton X-100, 0.1% sodium lauryl sarcosine, 10 mMbeta-mercaptoethanol (extraction buffer) by freezing with liquidnitrogen and grinding with mortar and pestle with sand or glass beads.Disposable pestles that fit into Eppendorf tubes (Kontes Glass) proveduseful for homogenizing small bits of tissue (e.g. leaf). Extracts canbe stored at -70° C. with no loss of activity for at least two months.Storage of extracts in this buffer at -20° C. should be avoided, as itseems to inactivate the enzyme.

8.1.6. Spectrophotometric Assay

Reaction buffer was 50 mM NaPO₄ pH 7.0, 10 mM beta-mercaptoethanol, 1 mMEDTA, 1 mM p-nitrophenyl glucuronide, 0.1% Triton X-100 in 1 ml.reaction volumes, and incubated at 37° C. The reactions were terminatedby the addition of 0.4 ml of 2-amino, 2-methyl propanediol (SigmaA-9754). Absorbance was measured at 415 nm against a substrate blank.Under these conditions the molar extinction coefficient of p-nitrophenolis assumed to be 14,000, thus in the 1.4 ml final volume, an absorbanceof 0.010 represented about one nanomole of product produced. One unit isdefined as the amount of enzyme that produces one manomole ofproduct/minute at 37° C. This would represent about 5 ng of purebeta-glucuronidase.

8.1.7. Fluorometric Assay

The fluorogenic reactions were carried out in 1 mM 4-methyl umbelliferylglucuronide in extraction buffer with a reaction volume of 1 ml. Thereactions were incubated at 37° C., and 200 μl aliquots were removed atzero time and at subsequent times and the reaction terminated with theaddition of 0.8 ml 0.2M Na₂ CO₃. The addition of Na₂ CO₃ served the dualpurposes of stopping the enzyme reaction and developing the fluorescenceof MU, which is about seven times as intense at alkaline pH.Fluorescence was then measured with excitation at 365 nm, emission at455 nm on a Kontron SFM 25 Spectrofluorimeter, with slit widths set at10 nm. The resulting slope at MU fluorescence versus time couldtherefore be measured independently of the intrinsic fluorescence of theextract. The fluorometer was calibrated with freshly prepared 4-methylumbelliferone (MU) standards of 100 nanomolar and 1 micromolar MU in thesame buffers. Fluorescence was linear from nearly as low as the machinecan measure (usually 1 nanomolar or less) up to 5-10 micromolar 4-methylumbelliferone.

Protein concentrations of plant extracts and a purifiedbeta-glucuronidase were determined by the dye-binding method of Bradford(Bradford, 1976, Anal. Biochem. 72:248), with a kit supplied by BIO-RADLaboratories.

DNA concentrations in extracts were determined by measuring thefluorescence enhancement of Hoechst 33258 dye (Laborca and Paigen, 1980,Anal. Biochem. 102:3434-352), with the calibrations performed byaddition of lambda DNA standards to the extract to eliminate quenchingartifacts.

8.1.8. In Situ Localization of GUS Activity In SDS Polyacrylamide Gels

Plant extracts (1-50 μl ) were incubated with 2 volumes of SDS Samplebuffer at room temperature for approximately 10-15 minutes and thenelectrophoresed on a 7.5% acrylamide SDS gel (Laemmli, 1970, Nature227:680) overnight at 50 mA, or in a mini-gel apparatus (BIO-RAD) for 45minutes. The gel was then rinsed 4 times with gentle agitation, in 100ml extraction buffer for a total of 2 hours, incubated on ice in assaybuffer (containing MUG) for 30 minutes, then transferred to a glassplate at 37° C. After approximately 10-30 minutes at 37° C., dependingon the sensitivity required the gel was sprayed lightly with 0.2M Na₂CO₃ and observed under long wavelength UV transillumination. Gels werephotographed using a Kodak 2E Wratten filter.

8.1.9. Histochemical Assay

Sections were cut by hand from unfixed stems of plants grown in vitroessentially as described in O'Brien et al. (1981, in "The Study of PlantStructure: Principles and Selected Methods," Termarcarphi Ptx LtdMelbourne, Australia) and fixed in 0.3% formaldehyde in 10 mM pH 5.6,0.3M mannitol for 45 minutes at room temperature, followed by severalwashes in 50 mM NaH₂ PO₄, pH 7.0. All fixatives and substrate solutionswere introduced into interstices of sections with a brief (about Iminute) vacuum infiltration. A good review of histochemical techniquesand the caveats to their utilization and interpretation can be found inPearse (1972, in "Histochemistry: Theoretical and Applied, ThirdEdition, Vol. II, Churchill Livingstone, Edinburgh, pp. 808-840).Substrates for histochemical localization include indigogenic dye5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc), and napthol ASBIglucuronide.

Histochemical reactions with the indigogenic substrate,5-bromo-4-chloro-3-glucuronide (X-gluc) were performed with 1 mMsubstrate in 50 mM NaH₂ PO₄ pH 7.0 at 37° C. for times ranging from 20minutes to several hours. After staining, sections were rinsed in 70%ethanol for 5 minutes, then mounted for microscopy.

Cleavage of napthol ASBI glucuronide releases the very insoluble freenapthol ASBI which is either simultaneously coupled, or post-coupledwith a diazo dye to give a colored product at the site of enzymeactivity. Post-coupling is preferred, as it seems to give a much lowerbackground. Sections were incubated in 0.1M NaH₂ PO₄ pH 7.0 with 1 mMNapthol ASBI glucuronide in a moist chamber at 37° C. for 15 minutes to3 hours. The specimen was then washed in phosphate buffer and coupledusing a fresh solution of diazotized dye in phosphate buffer. Post-coupling was performed with a 1-3 mg/ml solution of Fast Garnet GBC inphosphate buffer, pH 7, after which the sections were washed and mountedfor light microscopy.

Fixation was accomplished with 2.5% glutaraldehyde in 0.1M NaH₂ PO₄ pH7.0 for 2-3 minutes on ice.

8.1.10. Purification of Beta-Glucuronidase

Beta-glucuronidase was purified essentially as described from E. colicells containing the plasmid pRAJ210. Eight liters of cells were grownin L broth with 50 ug/ml ampicillin at 37° C. with vigorous agitation.The cells were harvested as they approached saturation, washed in M9salts, and resuspended in about 100 ml of 100 mM NaH₂ PO₄ pH 7.0 10 mMbeta-mercaptoethanol, 50 mM NaCl, 0.2% Triton X-100 and 25 ug/mlphenylmethyl sulphonyl fluoride. The slurry was passed through a Frenchpressure cell at 12,000 psi, and the resultant lysate was stirred on icefor 30 minutes. The lysate was spun at 10,000×g for 30 minutes at 4° C.,and the turbid supernatant was dialysed overnight against severalchanges of 50 mM Tris pH 7.6, 10 mM B-ME (buffer A). The dialysate wasloaded onto a column of DEAE Sephacelo (2.5×40 cm) equilibrated in thesame buffer, at 4° C. The column was washed with loading buffer andeluted with a 500 ml linear gradient of NaCl (0-0.4M). The combined peakfractions were concentrated in an Amicon ultrafiltration apparatus witha PM 30 membrane to a final volume of 27 ml. This volume was loaded ontoa 500 ml (2.5×100 cm) Sephacryl S-200 gel filtration column, and elutedwith buffer A plus 100 mM NaCl. Peak fractions were pooled and dialysedovernight against 20 mM NaOAc pH 5.0, at 4° C. The resulting pellet wasdissolved in buffer A, and both pellet and supernatant were assayed forbeta-glucuronidase activity and analyzed by SDS-PAGE. The supernatantcontained the majority of the activity, and by gel analysis had lostnearly all of the contaminating protein (greater than 95% purity asjudged by Coomassie staining) obviating further chromatography. Thepurified enzyme was stored in GUS extraction buffer at 4° C. The finalyield was about 350 mg.

8.2. Results

8.2.1. Higher Plants Contain No Detectable Beta-Glucuronidase Activity

Roots, stems and leaves were taken from wheat, tobacco, tomato, potato,Brassica napus and Arabidopsis thaliana. Potato tubers and seeds fromwheat and tobacco were homogenized with GUS extraction buffer containinga variety of protease inhibitors such as PMSF and leupeptin. The plantextracts were incubated in a standard assay at 37° C. for 4 to 16 hours,and the fluorescence of MU was measured. Endogenous activity was belowthe limits of detection. Extremely lengthy assays occasionally gave lowlevels MU fluorescence, but the kinetics of MU accumulation wereconsistent with a slow conversion of the glucuronide into another form,possibly a glucoside, that was subsequently cleaved by intrinsicglycosidases. Beta-galactosidase assays performed under similarconditions on tobacco and potato extracts were off-scale (at least10,000 times higher than the minimal detectable signal) within 30minutes. Reconstruction experiments were performed with purified GUSadded to tobacco and potato extracts to demonstrate the ability of theseextracts to support beta-glucuronidase activity.

8.2.2. Construction of Plasmids for Transformation of Plants with GUSFusions

A general purpose vector for constructing gene fusions was made byligating the coding region of GUS 5' of the nopaline synthasepolyadenylation site (Bevan et al., 1983, Nature 304:184-187) in thepolylinker of pBIN 19. This vector, pBI101 (see FIG. 9) contains uniquerestriction sides for Hind III, Sal I, Xba I, BamH I, and Sma I upstreamof the AUG initiator codon of GUS, to which promoter DNA fragments canbe conveniently ligated. The cauliflower mosaic virus 35S promoter(O'Dell et al., 1985, Nature 313:810-812) in the expression vector pROK1(Baulcombe et al., 1986, Nature 321:446-449) was ligated into the HindIII and BamH I sites to create pBI121. Similarly the promoter from atobacco gene encoding the small subunit of ribulosebisphosphatecarboxylase small, Ntss23 (Mazur and Chui, 1985, Nucl. Acids Res.13:2373-2386) deleted of rbcS coding sequences was fused to pBI101 tomake pBI131.

FIG. 9 illustrates the structure of the expression vectors.

The lower portion of FIG. 9 shows the T-DNA region of pBI101, containingpolylinker cloning sites upstream of the beta-glucuronidase gene,followed by the nopaline synthase polyadenylation site (NOS-ter). Pst Iand Sph I are not unique to the polylinker. The expression cassette iswithin pBIN 19, giving pBI101 a total length of approximately 12 kb.

The middle portion of FIG. 9 shows chimeric CaMV 35S-GUS gene in pBI121.An 800 bp Hind III--BamH I CaMV 35S promoter fragment (Guilley et al.,1982, Cell 30:763-773) was ligated into the corresponding sites ofpBI101. The mRNA initiation site is approximately 20 bp 5' of the GUSinitiator codon.

The top portion of FIG. 9 shows chimeric rbcS-GUS gene in pBI101. A 1020bp Hind III--Sma 1 fragment containing the promoter of a tobaccoribulose bisphosphate caraboxylase small subunit gene (rbcS) was ligatedinto the corresponding sites of pBI101. The mRNA initiation site isapproximately 55 bp 5' of the GUS initiator codon, and contains nearlythe entire untranslated leader of the rbcS gene.

FIG. 9 also illustrates the differences between the three pBI101plasmids.

8.2.3. Chimeric GUS Genes are Expressed in Transformed Plants

Nicotiana tabacum var. Samsun plants were transformed with Agrobacteriumbinary vectors (Bevan, 1984, Nucl. Acids Res 12: 8711-8721) containingtranscriptional fusions of either the CaMV 35S promoter or the tobaccorbcS promoter with the coding region of GUS as shown in FIG. 9. Severalkanamycin resistant plants were regenerated from each transformation.

Two rbcS-GUS transformants and two CaMV-GUS transformants were chosenfor further study. First assays were made of various organs of one plantfrom each transformation, axenically cultured in 300 lux white light, 18hour day, 6 hour night. Extracts were prepared from axenic tobaccoplants using about 50 mg fresh weight of tissue ground in 500 ulextraction buffer. 5 μl of extract was assayed in 500 ul extractionbuffer. 5 μl of extract was assayed as described in "Materials andMethods" above. Mature leaves were lower, expanded leaves approximately80 mm long, while young leaves were approximately 5 mm long, and weredissected from the shoot apex. All samples were taken from the sameplant (either CaMV-GUS 21, rbcS GUS 2 or non-transformed) at the sametime. Leaf tissue was taken from a non-transformed plant for this assay,although all organs showed no GUS activity.

The results of this analysis are shown in FIG. 10, and tabulated inTable II using either of two normalization methods (see followingdiscussion).

                  TABLE II                                                        ______________________________________                                        GUS Specific Activity                                                                                 (pmoles                                                       (pmoles         4-MU/min/mg                                                   4-MU/min/mg protein)                                                                          DNA)                                                  Gene Fusion:                                                                            CaMV     rbcS-   untrans-                                                                             CaMV   rbcS-                                Plant organ                                                                             35S-GUS  GUS     formed 35S-GUS                                                                              GUS                                  ______________________________________                                        Leaf (5 mm)                                                                             283        205   <0.1    2,530  4,400                               Leaf (70 mm)                                                                            321      1,523   <0.1    5,690 93,950                               Stem      427        260   <0.1   13,510  2,650                               Root      577        62    <0.1   12,590   690                                ______________________________________                                    

The rate data shown in FIG. 10 were converted to specific activity bymeasuring the protein concentration of the extracts using the Bradfordreagent. The data are also presented as GUS activity per unit weight ofDNA in the extract to better account for the differences in cell numberbetween different tissues.

The plant containing a rbcS-GUS fusion (rbcS-GUS 2) exhibited a patternof gene expression consistent with earlier studies using heterologousrbcS gene fusions (Simpson et al., 1986, Nature 323:551-554). Thehighest specific activity, using either protein or DNA as a denominator,was found in older leaves (about 8 cm long), with progressively lessactivity in very young leaves 5(less than 5 mm), stems and roots. Theother rbcS-GUS fusion plant showed a similar pattern.

The two plants transformed with the CaMV 35S-GUS fusion displayed apattern of gene expression distinct from that of the rbcS-GUS fusionplants. The highest levels of activity were found in roots, with similarlevels in stems. GUS activity was also high in leaves, consistent withprevious observations that the CaMV 35S promoter is expressed in allplant organs (O'Dell et al., 1985, Nature 313:810-812).

To verify that no significant rearrangements of the transforming DNA hadoccurred, a Southern blot analysis was conducted as shown in FIG. 11,which is an autoradiograph of a Southern blot of DNA extracted fromtransformed plants and digested with restriction endonucleases. Thefilter was hybridized with a ³² p labelled restriction fragmentcontaining the coding region of the beta-glucuronidase gene. In thisFigure,

Lane 1. CaMV-GUS 21 EcoR I

Lane 2. CaMV-GUS 21 EcoR I & Hind III

Lane 3. CaMV-GUS 29 EcoR I

Lane 4. CaMV-GUS 29 EcoR I & Hind III

Lane 5. rbcS-GUS 2 EcoR I

Lane 6. rbcS-GUS 2 EcoR I & Hind III

Lane 7. rbcS-GUS 5 EcoR I

Lane 8. rbcS-GUS 5 EcoR I & Hind III

Lane 9. Non-transformed EcoR I

Lane 10. Non-transformed EcoR I & Hind III

Lane 11. Single copy reconstruction of GUS coding region

Lane 12. Five copy reconstruction.

Digestion of DNA extracted from all of the transformants with Hind IIIand EcoR I released a single internal fragment of T-DNA consisting ofthe nopaline synthase polyadenylation site, the GUS coding region andthe promoter (CaMV35S or rbcS). RbcS-GUS transformants contained 3copies (rbcS-GUS 2, FIG. 11, lane 6) and about 7 copies (rbcS-GUS 5,lane 8) of the predicted 3.1 kb Hind III--EcoR I fragment. Digestionwith EcoR I revealed multiple border fragments (FIG. 11, lanes 5 and 7)confirming the copy number estimates deduced from the double digestions.Similarly CaMV 35S-GUS plants had multiple insertions as shown in FIG.11 lanes 1 to 4. CaMV-GUS 21 had 3 copies of the predicted 2.9 kbfragment, while CaMV-GUS 29 had 2 copies. No hybridization of thelabelled GUS coding region to untransfomred plant tissue was observed(lanes 9 and 10).

8.2.4. Visualization of GUS Activity on SDS-Polyacrylamide Gels

Extracts of transformed plants were prepared and electrophoresed,together with negative controls and varying amounts of purifiedbeta-glucuronidase, on an SDS-polyacrylamide gel. The gel was rinsed toreduce the SDS concentration, and then treated with the fluorogenicsubstrate, MUG. After the reaction had progressed sufficiently, the gelwas made alkaline to enhance fluorescence, placed on a long wave UV boxand photographed (FIG. 12). The gel was trans-illuminated with 365 nmlight and photographed using a Kodak Wratten 2E filter. In FIG. 12,

Lane 1. Transformed plant extract--CAB-GUS fusion

Lane 2. Transformed plant extract--SSU-GUS 2

Lane 3. Transformed plant extract--CaMV-GUS 21

Lane 4. Non-transformed plant extract

Lane 5. Non-transformed plant extract plus 1 ng GUS

Lane 6. Non-transformed plant extract plus 10 ng GUS

Lane 7. Non-transformed plant extract plus 50 ng GUS

GUS activity could be seen in all lanes containing purified enzyme, witha limit of sensitivity in this experiment of one nanogram. In otherexperiments, we have observed activity with as little as 0.2 ng. Thelanes containing the SSU-GUS and CaMV-GUS fusion extracts show GUSactivity that migrates with the same mobility as the purified enzyme,indicating that translation is initiating and terminating at the correctlocations in the GUS sequence and that no significant post-translationalprocessing is occurring. An additional lane was included that containeda protein fusion between part of the tobacco chlorophyll a/b bindingprotein and GUS that shows decreased mobility relative to purifiedbeta-glucuronidase, as predicted. Staining of gels using thehistochemical methods described below proved to be effective, but not assensitive as the fluorogenic stain.

8.2.5. GUS Activity in Plants can be Visualized Using HistochemicalMethods

Although there are very few organs in plants, each organ is composed ofmany different cell types, often associated in the form of distincttissues. Since different organs consist of unequal combinations of thesecell types intermingled in a highly complex fashion, the meaningfulinterpretation of "organic-specific" gene expression becomes a difficultexercise. One approach to characterizing cell-type specific expressionof chimeric genes in plants has utilized microdissection (Simpson etal., 1986, Nature 323:551-554). These methods are, however, extremelylaborious, prone to varying degrees of contamination, and manycell-types within plants are inaccessible to the techniques.Alternatively, localization of chimeric gene activity by histochemicalmethods has been successful in other systems (Lis et al. 1984, Cell35:403-410).

To determine whether it would be possible to use histochemistry toinvestigate single-cell or tissue-specific expression of GUS genefusions in plants, preliminary experiments were carried out on sectionsof stems of several independently transformed rbcS-GUS and CaMV-GUSplants. Stem sections were chosen both for their ease of manipulationand because most of the cell types of a mature plant are represented instem. To illustrate the light-regulated nature of the rbcS-GUS fusion,the plants were illuminated from one side only for one week beforesectioning. Sections from both plants stained intensely with thesubstrate while non-transformed tissue did not stain. Sections ofCaMV-GUS plants always show highest levels of activity in phloem tissuesalong the inside and outside of the vascular ring, most prominently in apunctate pattern that overlies the internal phloem and in the rays ofthe phloem parenchyma which join the internal and external phloem (Esau,19787, The Anatomy of Seed Plants). There is also variable lighterstaining throughout the parenchymal cells in the cortex and in the pith,and also in the epidermal cells, including the trichomes.

RbcS-GUS stem sections rarely, if ever, show intense staining in thetrichomes, epidermis, vascular cells or pith, but tend to stain mostintensely over the cortical parenchyma cells containing chloroplasts(chlorenchyma), with faint and variable staining in the pith. Althoughthe strongest staining is most often seen in a symmetrical ring aroundthe vascular tissue just inside the epidermis, an asymmetricdistribution of staining in the cortical stem cells is sometimesobserved. Suspecting that this pattern was due to uneven lighting, aplant was illuminated from one side for one week before sectioning, andit was found that the staining was asymmetric, with intense staining inthe chloroplast-containing cells proximal to the light source. Thestaining patterns observed for both the CaMB 35S-GUS and the rbcS-GUStransformants are consistent between several independent transformants.Untransformed plants never show staining with X-Gluc, even afterextending assays of several days.

8.3. Discussion

New methods are provided for analyzing gene expression in transformedplants that are potentially of general utility. The beta-glucuronidasegene from E. coli has been expressed at high levels in transformedtobacco plants with no obvious ill effects on plant growth orreproduction. It should be emphasized that the determination of rates ofenzyme activity allows accurate determination of quantity of chimericgene production, even over an intrinsically fluorescent background. Thefluorometric assay is very specific, extremely sensitive, inexpensiveand rapid. Minute quantities of tissue can be assayed with confidence;recently GUS levels have been measured in isolated single cells oftransformed plants.

Beta-glucuronidase is very stable in extracts and in cells, with ahalf-life in living mesophyll protoplasts of about 50 hours. Because ofthis, it is felt reasonable to interpret GUS levels as indicative of theintegral of transcription and translation, rather than the rate. Inaddition, GUS is not completely inactivated by SDS-PAGE, can toleratelarge amino-terminal fusions without loss of enzyme activity and can betransported across chloroplast membranes with high efficiency. It isbelieved, therefore, that the system will also be very useful instudying the transport and targeting of proteins, not only in plants,but in other systems that lack intrinsic beta-glucuronidase activity,such as Saccharomyces cerevisiae and Drosophila melanogaster.

A commercially available histochemical substrate has been used todemonstrate GUS activity in transformed plant tissue. Other substratesare available and give excellent results. It is emphasized thatmeaningful interpretation of results of histological analysis in termsof extent of chimeric gene activity, whether by in situ hybridizationmethods or by histochemistry, as presented here, is not a trivial orstraightforward matter. However, with these cautions, histochemicalmethods can be very powerful for resolving differences in geneexpression between individual cells and cell-types within tissue.

A distinctly non-uniform distribution of GUS activity in stem sectionsof several CaM-GUS transformed plants has been observed. Differentcell-types within plants are expected to have differing metabolicactivity with corresponding differences in rates of transcription andtranslation, and our results may reflect such a difference.Alternatively, since many of the cells of the phloem have very smallcross-sectional areas, the intense dye deposition seen in these regionsmay simply reflect the greater cell number per unit area. Thelocalization that is observed may also be due to a real difference inthe level of expression of the CaMV 35S promoter between cell types.Recently, it has been argued that the CaMV 35S promoter ispreferentially active in cells during the S phase of the cell cycle. Ifthis is true, then the pattern of GUS staining observed may reflect celldivision activity in these cells. This observation is consistent withthe proposed role of the 35S transcript of CaMV in viral replication(Pfeiffer and Hohn, 1983, Cell 33:781-790). It is also interesting thatthe other class of plant DNA viruses, the geminiviruses, replicates inthe phloem parenchyma (Kim et al., 1978, Virology 89:22-23). It isconcluded therefore that it is no longer adequate to describe the 35Spromoter as "constitutive" solely by the criteria of expression in allplant organs, when there may be a strong dependence of transcription oncell-type or cell cycle.

The distribution of GUS activity in the stem sections of plantstransformed with rbcS-GUS genes is consistent with data that indicate arequirement for mature chloroplasts for maximal transcription ofchimeric rbcS genes (Simpson et al., 1986, Nature 323:551-554). Corticalparenchymal cells in the stem contain varying numbers of chloroplasts,while those in the pith and epidermis of the stem rarely containchloroplasts.

Different cell-types present in each organ contribute differently to thepatterns of gene expression, and each organ consists of differentproportions of these cell-types. It has been undertaken to minimize thiseffect on quantitative analysis of extracts by suitable choice of adenominator. The parameter that needs to be studied with gene fusions ismost often the expression of the gene fusion in each cell. Whenpreparing homogenates from plant organs, the number of cells thatcontribute to the extract will vary, as will the protein content of eachcell and cell-type. The DNA content of the extract will reflect thenumber of cells that were lysed (Labarca et al., 1980, Anal. Biochem.102:344-352) whereas the traditional denominator, protein concentration,will not. For example, a single leaf mesophyll cell contains much moreprotein than a single epidermal cell or root cortical cell. However,each will have the same nucleus with the same potential to express theintegrated gene fusion.

Using this approach, it is found that the differential expression of therbcS-GUS fusion is much more pronounced between immature and mature leafwhen GUS activity is expressed, per mg of DNA (see Table II). Whenprotein concentration is used as a denominator, the massive induction ofGUS activity during leaf maturation is masked by the concomitantinduction of proteins involved in photosynthesis.

The observation that the specific activity of GUS produced by CaMV-GUSfusions is the same in immature and mature leaves when expressed using aprotein denominator indicates that the rate of GUS accumulation closelyfollows the rate of net protein accumulation. The two-fold difference inGUS specific activity using a DNA denominator illustrates theaccumulation of GUS per cell over time. This quantitative analysis,together with histochemical data, may indicate that the differencesbetween GUS activity in the leaf, stem and root of CaMV-GUS fusionplants could reflect the larger proportion of phloem-associated cells inroots and stems compared to leaves. It is felt that the choice of a DNAdenominator best reflects the expression per cell, and hence is a moreaccurate reflection of the true regulation of the gene.

9. EXAMPLE: CLONING AND EXPRESSION OF THE ESCHERICHIA COLI GLUCURONIDEPERMEASE GENE

Upon sequencing the gene for β-glucuronidase sequence, analysisindicated the presence of a second open reading frame of at least 340bp, whose initiator codon overlapped the translational terminator of theβ-glucuronidase gene. This open reading frame was found to betranslationally active (Jefferson, R. A., 1985, Dissertation, Universityof Colorado, Boulder).

9.1. Materials and Methods

9.1.1. Plasmids and DNA

FIG. 14 illustrates the clones used to analyze the uid A locus of E.coli. pRAJ220 plasmid was used to deduce the sequence ofβ-glucuronidase; pRAJ210 was subcloned and the fragments 3' to theβ-glucuronidase gene, encoding the open reading frame, were sequenced.The resulting DNA sequence and predicted protein are shown in FIG. 15.

9.2. Results and Discussion

9.2.1. Locating the Glucuronide Permease Coding Region

By analogy with existing operons of E. coli, it appeared that the openreading frame encoded a permease protein that could facilitate theuptake of β-glucuronides. The lactose and melibiose operons (forexample) consist of a gene encoding a hydrolytic enzyme followed by acocistronic gene for the corresponding transport protein, or permease.This format, of genes with interdependent functions being located on thesame mRNA and subject to the same controlling mechanisms, is ubiquitousin bacteria. Because the substrates for glucuronidase are very polar, itis certain that they require active transport across the bacterialmembrane. The level of genetic analysis performed on the uid locus wouldnot have distinguished a mutation that eliminated β-glucuronidasefunction from a mutation that eliminated transport of a substrate,consistent with tight linkage between β-glucuronidase and glucuronidepermease. These facts led to the hypothesis that the open reading frameencoded the glucuronide permease. Further analysis has also indicatedthat the range of substrates for β-glucuronidase that can be transportedby the glucuronide permease is much wider than that of any previouslydescribed glycoside permease.

9.2.2. Analysis of Amino Acid Sequence and the Glucuronide PermeaseProtein

The predicted amino acid sequence of the putative glucuronide permeasewas subjected to computer analysis to determine the existing sequencesto which it had closest homology. The only two sequences that hadsignificant homology to the glucuronide permease were the melB geneproduct, the melibiose permease, and the lacY gene product, the lactosepermease. Of these, the homology with the melibiose permease is thestrongest, and is shown in FIG. 16. Interestingly, both the melibioseand lactose permeases are members of a class of sugar transporters thatuse the proton gradient of the cell membrane to drive the transport ofthe sugar against a concentration gradient. These permeases are also inthe unusual class that have been purified and shown to be active assingle proteins, and whose activity can readily be reconstituted invitro by addition of the pure permease to membrane vesicles.

The deduced amino acid sequence for glucuronide permease was subjectedto a computer analysis to predict the structure of the protein. Suchresults are shown in FIG. 17. The salient feature of the analysis is theextremely hydrophobic nature of the protein, and the long stretches ofhydrophobic amino acids that could easily span a membrane. TheKyte-Doolittle predictions of hydropathy (shown in the bottom frame)reveal hydrophobic regions that are located at almost identicalpositions to those of the melibiose permease, and at very similarpositions to those of the lactose permease.

9.2.3. Molecular Genetic Demonstration of Glucuronidase Permease

The proof of the glucuronide permease function was obtained by cloningthe putative permease gene under the control of a heterologous promoter,in this case the promoter of the lactose operon of E. coli. Whenwild-type E. coli cells are planted on LB agar petri plates containingthe chromogenic substrate for GUS,5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (called X-Gluc), thecolonies remain white. When excess glucuronidase is present in thecells, for instance when encoded by a plasmid, the colony turns blue,due to deposition of the indigo dye. The blue color in this case iscaused by (GUS) enzyme that is released from the many broken cells inthe colony. This tends to give a relatively diffuse blue colony, withdye being deposited on the agar around the colony as well as on thecolony itself. If however, a plasmid containing, not GUS, but rather thepermease gene linked to the lac promoter, is introduced into thewild-type cells, the colonies on X-gluc become deep blue. The phenotypeis even more striking because the blue color is very discrete, and isstrictly localized to the colony. These colonies do not produce anydetectable GUS in the absence of x-gluc but rather are induced byproduce it when X-gluc is present. This is due to the X-gluc beingtransported into the cell, binding to the uidR gene product (therepressor of the uid operon) and allowing expression of GUS. Thisphenomenon requires the glucuronide permease action. This can best beseen in the series of cloning experiments summarized in Table III.

                  TABLE III                                                       ______________________________________                                        BEHAVIOR OF E. COLI STRAIN                                                    DH5-CONTAINING VARIOUS lacZ-GLUCURONIDE                                       PERMEASE FUSIONS IN pUC19                                                     Plasmid              Color on X-Gluc plates                                   ______________________________________                                        pRAJ 280 (lacZ/permease fusion)                                                                    Blue                                                     pRAJ 281 (Sst I lacZ frameshift)                                                                   Blue                                                     pRAJ 282 (Nsi I frameshift)                                                                        White                                                    pRAJ 283 (Acc I frameshift)                                                                        White                                                                         (trace of blue at 2 days)                                pRAJ 284 (Sst I & Ban II frameshift)                                                               Whiteish                                                                      (very pale blue)                                         pRAJ 285 (Nco I- Pst I 3' deletion)                                                                Blue                                                     pRAJ 286 (5' end, Bam HI linker)                                                                   Dark Blue (small)                                        pRAJ 287 (3' end Bam HI linker)                                                                    Dark Blue (small)                                        ______________________________________                                    

The glucuronide permease gene was subcloned into pUC19 to give a genefusion with lac that caused E. coli to give discrete blue colonies onX-gluc. This was then subjected to various changes to alter the readingframe of the predicted permease to determine whether the reading framewas required for the blue colony phenotype. Restriction endonucleasesites were chosen that were distributed throughout the gene. Some ofthese are indicated in FIG. 15. The restriction sites were cleaved andfilled in to mutate the area around the site by shifting the putativereading frame. Such a shift occurring upstream of the glucuronidepermease initiator codon (pRAJ218) showed no change in the color of theresulting colony. However, frame shifts within the coding sequenceeliminated or severely reduced the capacity of the gene to give rise tocolored colonies. In particular the Nsi I site mutant (PRAJ282) wascompletely colorless and the Acc I site mutant (pRAJ283) was almostcompletely colorless, with just a trace of blue after two days. The BanII site frame-shift showed a faint trace of blue overnight with anobvious, but still quite pale, blue after two days on plates. Theelimination of all blue color by the Nsi I mutant is expected, as theamount of permease made before the frame shift is very small--on theorder of 100 amino acids. The next frame shift, the Acc I mutant, showeda trace of permease action. This may be because the amount of permeasemade (more like half of the permease) could have residual activity. TheBan II mutant (in which more than 80% of the permease is made) shows adefinite but severely reduced activity. This is also as would bepredicted, and demonstrates conclusively the role of the open readingframe in the development of the blue colony.

The deletion mutant that extended 3' from the Nco site at bp 1510(pRAJ285) caused no obvious change, leaving dark blue colonies. Thisverified the 3' extent of the gene as predicted by DNA sequencing. Thecontext of the start site of the gene was altered by oligonucleotidemutagenesis in order to verify its location. This resulted in a permeasegene deleted of sequences up to -12 from the initiator codon. Thismutant showed an even darker blue colony (and smaller--presumably due tothe over-expression of the membrane protein). The higher level ofpermease in these cells may be due to better translation on the newmRNA, perhaps because of loss of attenuating sequences. This clone,pRAJ286, contains a Bam HI linker immediately 5' of the Shine/Delgarnosequence and ensures that the initiator codon is the first one presentedon any hybrid mRNA produced from this cloned fragment. To make a moreuseful cassette, pRAJ286 was modified by the addition of another Bam HIlinker at the 3' end. This vector, pRAJ287, contains the entireglucuronide permease gene as a Bam HI fragment within the polylinkersites of the plasmid pUC19.

Next, the ability of the glucuronide permease to transport substratesother than X-gluc was tested. If the permease could transport such alarge heterocyclic molecule as X-gluc, it was reasonable that it couldtransport other complex glucuronides, and hence offer a general route totransporting GUS substrates. Two bacterial cultures, one containing theplasmid pRAJ230 and the other pRAJ210 (Jeffeson et al. 1986) were grownto similar densities in L broth. Both these cultures produce GUS withinthe cells. pRAJ210 also includes the DNA encoding the permease--pRAJ230is deleted of most of the permease gene. The cultures were washedextensively to eliminate GUS from the medium, and incubated with asolution of 4-methyl-umbelliferyl glucuronide, a fluorogenic substratefor GUS. The culture containing PRAJ210 immediately began to fluoresceintensely, while the culture containing pRAJ210 did not. When thecultures were lysed with a sonicator in the presence of fluorogenicsubstrate, both extracts showed intense fluorescence indicative ofintact β-glucuronidase activity.

10. EXAMPLE: TRANSGENIC PLANTS EXPRESSING A BETA-GLUCURONIDASE GENEFUSION AND ALTERATION OF GROWTH PATTERNS BY AUXIN-GLUCURONIDE

10.1. Materials and Methods

Tryptophyl glucuronide was synthesized from indole-3-ethanol byconventional Koenigs-Knorr condensation. Leaf discs were prepared fromuntransformed plants or plants transformed with a highly expressed CaMV35S/GUS gene fusion as described in section 8, supra. Leaf discs fromnontransformed and CaMV 35S/GUS transformed plants were exposed to mediacontaining cytokinin and (i) no auxin, (ii) μM tryptophyl-glucuronide,or (iv) 10 μM tryptophyl glucuronide.

10.2. Results and Discussion

Indole-3-ethanol (tryptophol) has been shown to be an auxin in severalsystems. It has been proposed that indole ethanol represents a bufferedstorage form of auxin, in a branch point leading fromindole-3-acetaldehyde (the immediate precursor of the known auxinindole-3-acetic acid (IAA). In the absence of auxin, leaf discs fromcontrol plants and CaMV 35S/GUS-transformed "GUS-plants" becamechlorotic and died over a 7 week period (FIG. 18). In bothGUS-expressing and control plants, the effect of 1 μM IAA was similar,resulting in the persistance of chlorophyll and the maintenance ofliving healthy cells. On media in which tryptophyl glucuronide was thesole auxin source, only those leaves which expressed GUS remained greenand healthy, presumably because hey were able to cleave active auxinfrom tryptophyl glucuronide.

11. EXAMPLE: THE USE OF GUS FUSIONS IN TRANSGENIC PLANTS: REGULATION OFCHIMERIC PATATIN GENES IN TRANSGENIC POTATO PLANTS

While the potato is perhaps the most important non-cereal stable crop inthe world, at least in terms of acreage under cultivation and tonnage,it is one of the most poorly understood. The potato is a relatively newcrop, with only a modest history of genetic improvement when compared tocereals such as wheat and maize. Potatoes are propagated vegetatively,and most commercial cultivars are poorly characterized tetraploids oflimited fertility, hence conventional breeding is not simple orstraightforward. These factors strengthen the argument that potatoeswill be one of the first crops to specifically benefit from improvementby non-traditional means, and in particular by genetic engineering. As asolanaceous plant, Solanum ruberosum is highly susceptible to infectionby Agrobacterium tumefaciens, and genetic transformation by this routehas become routine. However, the ability to introduce new genes into acrop only makes more urgent our need to Understand how these genesfunction, so that sensible strategies for copy improvement by thesepowerful new tools can be developed.

The goal of the experiments described below was to develop anunderstanding of the behaviour and regulation of a gene encodingpatatin--a major protein of the potato tuber--in laboratory, glasshouseand field grown potato plants. Towards this end, transgenic approachesusing GUS fusions were used to increase the resolution of the analysisand to facilitate the experiments.

Patatin is a 40 kDa glycoprotein that accumulates in tubers to up to 40%of the total soluble protein (reviewed by Park, 1986 Potato Physiology,Li. ed., Academic Press). The function of patatin is still obscure, butit is clearly associated with storage tissue. Induction of patatin mRNAtranscription can also be observed in vitro when single node cuttingsare subjected to tuberization conditions, even when tuberization itselfdoes not occur (Paiva et al., 1983, Plant Physiol. 71:616-618). It isencoded by a gene family with more than a dozen members, meaning that inthe normally tetraploid cultivated potato there are upwards of fiftypossible genes bearing patatin coding sequences in many allelic andnon-allelic loci (Mignery et al., 1988 Gene 62:27-44; Twell and Ooms,1988 Mol. Gen. Genet. 212:325-326). Each of these genes may behavedifferently, some regulated to express at high levels, others at verylow or even inactive levels--and, perhaps, with different spatial andtemporal control regimes. How then can one distinguish between thecontribution of one gene from that of another, or even its allele? Theeasiest way, and the way that allows experimental manipulation of theDNA, is to mark one isolated gene in vitro such that it can be readilystudied distinct from others, and re-introduce it into the host genome;that is, to construct a transgenic plant using a chimeric gene, or genefusion.

11.1. Materials and Methods

To investigate the transcriptional regulation of the patatin gene intransgenic potato, simple gene fusions were generated between a promoterfrom a patatin gene and GUS. The patatin gene that was used was clonedand sequenced by Mike Bevan and his colleagues at the Plant BreedingInstitute using a cDNA probe obtained from Bill Park at Texas A&M. Theinitial DNA constructions were simple transcriptional fusions in whichvarying lengths of the 5' upstream regions of the patatin gene werefused to the coding sequence of GUS contained on a plasmid calledpRAJ260. Some of these fusions are outlined in FIG. 16. Theseconstructions were subcloned within a binary Agrobacterium vector,pBIN19 (Bevan, 1984, nucl. Acids Res. 12:8711-8721) and transformed intoa commercial potato cultivar by a tuber-disc method (Sheerman and Bevan,1987 Plant Cell Reports 7:13-16), selecting for resistance to theantibiotic kanamycin. Many independent transformants were obtained foreach of the promoter lengths, and subjected to further analysis.

11.2. Results and Discussion

11.2.1. In Vitro Induction Experiments

The first experiments carried out on the transformants were in vitroinductions of patatin-GUS activity. It had previously been shown byPaiva et al., (1983, Plant Physiol. 71:616-618) that accumulation ofpatatin protein could be induced in cuttings. To determine whether ourpatatin-GUS fusions were transcriptionally regulated accordingly, smallsingle-node cuttings of the primary transformants (approximately 100)were placed on media containing either 3% sucrose without cytokinin or7% sucrose with 2 mg/l benzyladenine, under either normal lightingregime or in the dark. Under these conditions,.about 30-60% of thecuttings in the dark gave small microtubers within 2 weeks, while in thelight the induced cuttings accumulated high levels of starch but showedno signs of tuberization. After two weeks incubation on either of thetwo media, the cuttings were homogenized using mortar and pestle, andGUS levels were determined with a fluorogenic assay. Most transformantsfrom each of the promoter classes showed strong induction of GUSactivity under conditions of high sucrose and cytokinin. Interestingly,GUS levels were highly elevated in the induced cuttings irrespective ofwhether they were grown in the light, or in the dark with concommitanttuberization. This observation is consistent with the experiments ofPaiva et al. (1983, supra) and Bourque et al., (1987, In Vitro Cell,Devel. Biol. 23:381-386) who showed that patatin synthesis could beuncoupled from tuberization. These experiments demonstrated clearly thatour patatin promoter would respond to induction phenomena that were insome sense distinct from those for tuberization--representing a subsetof the tuberization induction conditions. An alternative way tointerpret this is that patatin-GUS fusions are induced by all the sameconditions as those for tuberizations, but do not respond to a "normal"light-inhibition of tuberization.

To better define the nature of the induction signal, different sugarsand osmotica were used, including fructose, glucose, and mannitol, butnone showed a significant induction; sucrose alone gave high GUSactivity. In subsequent experiments we observed no reproduciblestimulation of patatin-GUS induction by cytokinin so it was omitted.

An example of the induction data obtained is shown in Table IV. Thistable shows the GUS levels accumulated in a cutting grown in vitro fortwo weeks under non-induced conditions, or under conditions that inducedpatatin with or without concommitant tuberization (dark and lightinduced, respectively). The data shown are for a single patatin-GUSdeletion derivative consisting of a 2164 bp promoter fragment directingGUS expression.

                  TABLE IV                                                        ______________________________________                                                 Induction Conditions                                                                          Fold Induction                                       Transformant                                                                             Uninduced Dark    Light Dark  Light                                ______________________________________                                        pBI141.3   1       15     40   160   3     10                                            #2.sup.  6    170   650   28    108                                           3       12     85   135   7     11                                            4       10     25   250   3     25                                            5        1     75    60   75    60                                            6        7     35   250   5     35                                            7        6     9    300   2     50                                            8        1     10    70   10    70                                            9       60     12   250     0.2  4                                            10       7     70   150   10    21                                            11      40    900   1700  22    42                                            #12 .sup.                                                                             15    150   3000  10    200                                           13       4     60   700   15    175                                           14       9     6    700     0.7 78                                            15      10    350   2300  35    230                                           16      40    320   1900  8     47                                            #17 .sup.                                                                             20    6700  7300  335   365                                Average n = 17     15    530   1169  33    90                                 Minimum             1     6     60     0.2  4                                 Maximum            60    6700  7300  335   365                                ______________________________________                                    

Analysis of Patatin-GUS Induction of Cuttings In Vitro for 17Independent Transformants With pBI141.4, Consisting of a 2164 bpPromoter Fusion to GUS. Cuttings were all approximately 1 cm long, witha single leaf node with no obvious auxiliary bud growth at time zero.Cuttings were placed in petri plates containing MS media plus 3% sucrose(c.), 7% sucrose & 2 mg/1 BAP (d. and e.) in the light (c. and e.) ordark (d.) for 14 days. The cuttings were removed and ground in a mortarand pestle and assayed for GUS fluorometrically. Values are expressed asnM MU/hr at 37° C. All values are per cutting, and not normalized. #denotes those plants chosen for statistical study and field trial.

While it is clear that this patatin promoter is fully capable ofinducing GUS under these conditions, it is also clear that there isvariation between individual transformants. Some of this variation maybe due to physiological differences in the cuttings and some must be dueto "position-effect" influences on the gene fusions. "Position-effect"is a very important but poorly defined issue in transgenic science thatrefers to variation in gene expression that can be ascribed to theinfluence of differing sites of integration of the foreign DNA. Allcurrently available methods for transformation of plants result inapparently random integration of the foreign DNA into the genome of thehost plant. This inability to target DNA to its homologous site or atthe very least reproducible site, and the resulting uncertainties causedby neighboring sequences, local structure constraints or even threedimensional positioning in the genome can result in a confusing andfrustrating variation in gene expression. The nature and mechanism(s) ofthis influence is not at all clear, nor is the extent of the influence.

To investigate the extent of variation that was not due to "positioneffect" but rather to other factors 72 clonal single node cuttings weretaken from a single set of identically maintained and aged in vitrogrown plantlets that were in turn all derived from a singletransformant. This transformanant, 141.4-17 had been propagated for atleast two years by nodal cuttings prior to analysis, and so wascertainly not chimeric. The cuttings were divided into two sets of 36cuttings, and placed on media containing either 3% sucrose or 8% sucrosein the light, noting from which region of the parental plant they werederived. After two weeks, they were homogenized and assayed for GUSactivity by fluorometric measurements. The results indicated that thevariation from one cutting to another was as high as one thousand-fold,although the means of the uninduced and induced cuttings were differentby a factor of ten. Moreover, the distribution of GUS activity among thecuttings was so broad that a third of the highest expressing uninducedcuttings overlapped a third of the lowest expressing induced cuttings.In addition, there was absolutely no correlation between the position ofthe cutting on the plant and the response of the cutting to induction.The scatter was such that it would easily be possible, by inadvertentlyselecting a small number of plants, to deduce that the patatin promoterwas repressed under conditions that clearly give rise, on the whole, toa high induction of transcription. When a particular transformant wasanalyzed at several different times--as close as a month apart or aslong as two years apart--the reproducibility of induction was very poor.While a highly active transformant (4-17 was our highest expressingtransformant) was usually very high, the actual extent of inductionvaried tremendously.

These observations suggested the possibility that the influence Of thephysiological state of the plant or cutting was contributing perhaps asmuch or more to the variation in patatin-GUS expression as the geneticcomponent of "position-effect". It is possible that the variation amongclonally propagated plants is either a characteristic of patatin generegulation, a general feature of gene regulation in sporophyticdevelopment, or indeed even a feature of our tissue culture conditions.It is easy to invoke models in which random, but stable methylationoccurs to promoter sequences during the propagation in tissue culturethat alters the ability of the gene fusion to express.

11.2.2. Patatin-GUS Expression in Planta

The next set of experiments was designed to investigate the temporalpatterns and spatial localization of patatin transcription--as measuredby GUS fusion activity--during the growth and development of the plant.A representative set of transformants--three from each promoter class,from 360 bp of 5' sequence to 3500 bp--were moved to glasshouseconditions in soil. The plants matured indistinguishably fromuntransformed control plants. Tubers, roots and aerial tissues wereassayed for GUS activity quantitatively with fluorogenic assays andhistochemically using the indigogenic substrate X-Glue(5-bromo-4-chloro-3-indolyl-β-D-glucuronide).

As with the cuttings induced in vitro, there was a very wide range ofvariation in GUS activity observed in planta, when assayedquantitatively. In spite of this, there was a very strong trend for GUSlevels to be consistently much greater in tubers than in other organs.The answers to all of these questions will, of course, vary from systemto system, but will be important for providing a solid understanding ofthe behaviour of genes in individuals and in populations. It is alsoimportant to acquire this information for the more pragmatic purposes ofdesigning agronomically useful programs that involve gene transfer.

11.2.3. Design of the Field Trial

The Patatin-GUS field trial can be considered as three separate trials,designed to address different aspects of the questions outlined above.The first trial, called GUS I was designed to measure the degree ofvariation in gene expression observed between independenttransformants--that is, the "genetic" component of variation in chimericgene expression. GUS II is designed to ask how much variation is notgenetic, but is due to other factors, such as environmental,physiological or stochastic influences. GUS III is designed to ask howpatatin-GUS expression--both quantitatively and qualitatively--changesduring the course of the growing season.

11.2.3.1. GUS I

To investigate the extent of variation in patatin gene expression thatcould be ascribed to "position-effect" or otherwise characteristicgenetic variation, a large collection of independent transformants wasused. These transformants contained one of four different patatin-GUSgene fusions and were not preselected other than for the expression ofdetectable levels of GUS. Each transformant was propagated vegetativelyin vitro by nodal cuttings to generate 6 clonal replicates. A total of71 independent transformants was used, giving a total of 426 transgenicplants. Control plants that were not transgenic, but that had beenregenerated from shoot cultures or tuber disc, were also used. The trialwas replicated in six block, each self-containing but internallyrandomized.

11.2.3.2. GUS II

To determine the distribution of patatin-GUS gene expression and measurethe extent of variation within a particular transformant, i.e. thatwhich is not caused by genetic differences between individuals, butrather by environmental, physiological or stochastic differences, weselected 12 independent transformed plants, three from each of the fourpromoter deletion classes. The plants were preselected based on easilymeasurable GUS activity and strong induction of patatin-GUS undertuberization conditions in vitro. These twelve transformants werepropagated and multiplied vegetatiely to give 36 clonal replicas of eachplant. The trial was replicated in 36 internally randomized blocks andincluded shoot culture and tuber-disc regenerant controls. Since sixadditional replicates of each of these plants was represented in GUS I,the total available replication was up to 42.

11.2.3.3. GUS III

To investigate the changes in patatin-GUS expression during the growingseason, and to determine whether differences in organ-specificityoccurred during growth, the same twelve pre-selected transformants as inGUS II were propagated in vitro to give eighteen replicate plants foreach. These eighteen replicates of twelve transformants plus controlswere distributed in six blocks, each containing three replicatesrandomized internally. Every two weeks during the growing season, one ofthese blocks was randomly selected and harvested for analysis of GUSactivity.

11.2.3.4. Containment Consideration

In the design of the field trial, and during its execution, attentionwas given to minimizing the possibility of movement of the transgenicplant material off-site according to the recommendations of the AdvisoryCouncil on Genetic Manipulation, and by Plant Breeding Institute SafetyCommittee. This entailed placing the test plot well away from any otherpotato plot, planting guard rows of untransformed plants, containingmaterial during harvest and processing, removal of flower buds toprevent pollen formation and potential spread, and destruction oftransgenic plant material after completion of the field trial. Inaddition, the trial plot was sterilized and allowed to lie fallow for anextended time after harvest. The trial was performed under MAFF licenseno. PHF 48A/114(57).

11.2.3.5. Planting, Growth and Harvest Procedure

Young potato plantlets derived from tissue culture were grown in peatpots in a glasshouse to a height of approximately 5-10 cm. and plantedin the test plot on Jun. 1, 1987. The planting date was chosen tominimize the possibility of a late frost, which would have deleteriousconsequences on young plantlets. The usual agricultural practice ofplanting seed tubers was not employed due to logistical constraints ofproducing the seed material, and time constraints. The validity of theoutcome was not compromised by this planting regime, as the purpose ofthe trial was not to compare yield or agronomic performance withpotatoes grown under normal practice, but to investigate variationwithin and between similarly propagated populations.

During growth of the potato plants, manual weeding and spraying withcommercial fungicides and aphidicides was undertaken at regularintervals, and the plants were regularly irrigated. Immediately beforeharvesting, vigour was scored for all plants.

Harvesting of GUS III plants was performed every two weeks during thegrowing season beginning with an initial harvest of July 28. GUS I & IIwere harvested over the period from October 14-23. Harvesting wascarried out using manual fork lifting. For GUS III, the entire plant,including aerial structures, root and tubers was harvested at the sametime to ensure that the relative GUS levels in each tissue were directlycomparable. For GUS I & II, aerial structures were harvested first,followed about one week later with the tuber and root samples. Thisprotocol was followed to minimize the possibility that the tuberpopulation would be contaminated by fungal spores or other diseaseagents that would decrease their suitability for storage and replantingin a subsequent trial. In addition, this method most closelyapproximates agricultural practice in which the aerial structures areremoved well before harvest.

11.2.3.6. Sampling and Assay of GUS Activity

The logistics of sampling and analysis were greatly enhanced by the useof microtitre plates, and apparatus designed to use the characteristic8×12 array. These included 1 ml polypropylene tubes in racks with themicrotiter array (Micronic), microtiter plate carriers for thecentrifuge, multi-well pipettors and microtiter plate absorption andfluorescence spectrometers (Titertek Multiskan Plus and Fluoroskan II).Software for direct reading of the microtiter plates, correction ofmis-labelled or incorrectly specified wells, data analysis andmanipulation was developed expressly for this study ("Plates"Micro-Manipulator) by David Wolfe.

11.2.3.7. Results from the Field Analysis

Results from the field trial showed that even when grown in the fieldunder conditions that approximate agricultural practice, the netexpression of the patatin promoter is highly variable. It must beremembered that the GUS protein, being reasonably stable, reflects anintegral of gene expression over the lifetime of the reporter enzyme,not the instantaneous levels of transcription at the moment of harvest.This property is quite useful to minimize the variation that must occurdaily if not hourly. The trend is still maintained that expression is byfar the highest in the tubers, relative to other organis, but theextreme quantitive variation (more than one hundred fold) can bemirrored by qualitative variation. The plants that are highest in thetuber are rarely those that are highest in aerial tissues. Hence, notonly is absolute expression level modulated among clones, but relativeexpression in different organs as well. A possible model to explain acomponent of this variation emerges in considering some of the resultsfrom GUS III together with the laboratory analyses.

Studies of the mean GUS specific activity in five organ samples (tuber,root, node, stem and leaf from up to nine plants carrying the same genefusion) showed that "organ-specificity" is completely dependent on whenthe plants are harvested. Early harvests show a high degree of tuberspecificity, while lifts in the middle of the growing season show anincreasing degeneracy in expression. The third, fourth and fifth harvestdates show very high level expression in all aerial tissues--in a largenumber of plants the stem and node levels are higher than tuberlevels--while at the final harvest the tuber levels are prominant. It isinteresting to note that the specific activity of GUS in the tubersremains roughly constant during the growing season. This implies thatthe accumulation of GUS protein closely follows the net accumulation oftotal protein in the tuber--thereby maintaining a constant ratio. Thisindicates that the stability of GUS during growth is very similar to theoverall stability of patatin, since patatin accounts for the highestproportion of soluble proteins in the tuber. GUS activity is therefore,a measure, or "reporter," of protein content in tubers. Therefore, GUSactivity in these transgenic plants may be used to serve as a generalindicator of patatin promoter activity.

12. EXAMPLE: THE ENZYMATIC ACTIVITY ASSAY OF THE β-GLUCURONIDASE INDUCEDBY VARIOUS GLUCURIONIDES

Bacterial WLJ1 and SΦ200 (Δ gus operon), were cultured in LB liquidmedium until the cell absorbence reached 0.5 at 600 nm. 100 μl of thecell suspension was inoculated into 1 ml of LB liquid medium in a largetest tube. 100 μl of 50° μg/ml of each glucuronide (0.5 mg in 1 mlNaPO₄, pH 7.0) was then added (200 μg/ml for pregnane dial glucuronide).The culture was then incubated at 37° C. with agitation (Rolling plateincubator) for 2 hours. The cell was sedimented in an Eppendorf tube bycentrifugating at 13 krpm for 1 minute. The cell was then washedthoroughly with M9 salt (twice) and resuspended in 0.5 ml ofglucuronidase extraction buffer containing 50 μg/ml of chloramphenicolto stop the further. glucuronidase synthesis. The enzymatic activity ofβ-glucuronidase encoded by the gusA in each induction was then assayedbased on the cleavage of its substrate, p-nitrophenyl--glucuronide(pNPG). Being cleaved by the β-glucuronidase, the aglycone released fromthe substrate has an absorbance peak at 405 nm. At zero time, 10 μlglucuronidase extraction of each induction was then spontaneouslytransferred into five of the 100 μl the enzyme assay buffer(glucuronidase extraction buffer containing 1 mM pNPG). The fiveenzymatic reaction for each induction were then stopped by adding 100 μl10.4M Na₂ CO₃ respectively at five minutes intervals. The enzymaticactivity of glucuronidase was represented by the aglycone absorbance at405 nm for each induction is shown in Table V. Equal amounts of cellswere initially used for each induction. The strain SΦ200 (Δ gus operon)was used as a negative control.

13. DEPOSIT OF MICROORGANISMS

The following microorganisms containing the indicated recombinantplasmids were deposited with the National Collection of Industrial andMurine Bacteria, Torrey Research Station, Aberdeen.

    ______________________________________                                                              NCIB                                                    Plasmid       Host    Accession No.                                           ______________________________________                                        pBI 101.1     E. coli 12353                                                   pBI 101.2     E. coli 12354                                                   pBI 101.3     E. coli 12355                                                   ______________________________________                                    

The present invention is not to be limited in scope by the genes andproteins exemplified or deposited microorganisms which are intended asbut single illustrations of one aspect of the invention. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying figures. Such modifications areintended to fall within the scope of the appended claims.

                                      TABLE V                                     __________________________________________________________________________    THE EXPRESSION OF THE GUS OPERON INDUCED BY GLUCURONIDES                                          WJL1                   Sφ200 (gus)                                        Duration (minutes)     Duration (minutes)                 Glucuronide (Mwt)   5    10   15   20  25  5   10  15  20  25                 __________________________________________________________________________    No inducer          -0.070                                                                             0.029                                                                              -0.002                                                                             0.011                                                                             0.037                                                                             -0.122                                                                            -0.115                                                                            -0.103                                                                            -0.121                                                                            -0.115             Phenyl glucuronide (270.2)                                                                        0.349                                                                              0.746                                                                              0.988                                                                              1.377                                                                             1.258                                                                             -0.068                                                                            -0.071                                                                            -0.062                                                                            -0.072                                                                            -0.054             P-Nitrophenyl-glucuronide (315.2)                                                                 0.177                                                                              0.403                                                                              0.621                                                                              0.782                                                                             0.782                                                                             -0.086                                                                            -0.068                                                                            -0.079                                                                            -0.068                                                                            -0.098             4-Methulumbelliferyl-glucuronide (352.3)                                                          0.447                                                                              0.776                                                                              1.054                                                                              1.310                                                                             1.262                                                                              0.013                                                                             0.008                                                                             0.018                                                                             0.018                                                                             0.020             X-Gluc (521.8)      0.398                                                                              0.649                                                                              0.870                                                                              1.363                                                                             1.169                                                                             -0.046                                                                            -0.042                                                                            -0.017                                                                            -0.060                                                                            -0.041             Tryptophol glucuronide (380.3)                                                                    0.715                                                                              0.995                                                                              1.209                                                                              2.115                                                                             1.169                                                                              0.019                                                                            -0.045                                                                            -0.033                                                                            -0.041                                                                            -0.029             O-aminophenyl glucuronide (285.3)                                                                 0.596                                                                              0.951                                                                              1.144                                                                              1.861                                                                             1.471                                                                             -0.020                                                                            -0.017                                                                            -0.024                                                                            -0.024                                                                            -0.005             CN-umbelliferone-glucuronide (338.28)                                                             0.416                                                                              0.712                                                                              0.977                                                                              1.155                                                                             1.389                                                                             -0.006                                                                            -0.010                                                                            -0.026                                                                            -0.016                                                                            -0.030             Hydroxyquinoline glucuronide (321.3)                                                              0.064                                                                              0.111                                                                              0.174                                                                              0.251                                                                             0.288                                                                             -0.055                                                                            -0.066                                                                            -0.048                                                                            -0.054                                                                            -0.076             Naphthol ASBI glucuronide (548.4)                                                                 -0.053                                                                             -0.020                                                                             0.014                                                                              0.054                                                                             0.091                                                                             -0.127                                                                            -0.116                                                                            -0.110                                                                            -0.107                                                                            -0.101             Phenolphthalein glucuronide (493.4)                                                               0.023                                                                              0.066                                                                              0.081                                                                              0.147                                                                             0.183                                                                             -0.075                                                                            -0.064                                                                            -0.062                                                                            -0.057                                                                            -0.057             Estriol-3-glucuronide (463.5)                                                                     -0.003                                                                             0.061                                                                              0.082                                                                              0.117                                                                             0.166                                                                             -0.090                                                                            -0.076                                                                            -0.070                                                                            -0.083                                                                            -0.082             Estriol-17-glucuronide (463.5)                                                                    0.110                                                                              0.151                                                                              0.172                                                                              0.211                                                                             0.257                                                                              0.017                                                                             0.013                                                                             0.015                                                                             0.016                                                                             0.013             Estrone-17-glucuronide (463.5)                                                                    0.030                                                                              0.079                                                                              0.105                                                                              0.146                                                                             0.205                                                                             -0.050                                                                            -0.046                                                                            -0.046                                                                            -0.045                                                                            -0.045             Testorterone-glucuronide (463.5)                                                                  0.031                                                                              0.105                                                                              0.095                                                                              0.153                                                                             0.200                                                                             -0.045                                                                            -0.020                                                                            -0.042                                                                            -0.027                                                                            -0.034             Pregnane diol glucuronide (496.6)                                                                 0.035                                                                              0.066                                                                              0.100                                                                              0.134                                                                             0.165                                                                             -0.059                                                                            -0.063                                                                            -0.048                                                                            -0.060                                                                            -0.075             __________________________________________________________________________

What is claimed is:
 1. A transformed host cell containing a geneencoding beta-glucuronidase and a recombinant DNA molecule encoding theamino acid sequence of glucuronide permease as depicted in FIG. 15, or aDNA molecule that hybridizes under stringent conditions to thecomplement of the nucleotide coding sequence depicted in FIG. 15 andwhich encodes a functional glucuronide permease.
 2. The transformed hostcell of claim 1 in which the DNA encoding glucuronide permease has thenucleotide sequence as depicted in FIG. 15 from about nucleotide number94 to about nucleotide
 1461. 3. The transformed host cell of claim 1 inwhich the DNA encoding glucuronide permease has the nucleotide sequenceas depicted in FIG. 15 from about nucleotide number 106 to aboutnucleotide
 1461. 4. The transformed host cell of claim 1 in which theglucuronide permease ΔNA is under the control of a second nucleotidesequence that regulates gene expression in the host cell so thatglucuronide permease is expressed in the transformed host cell.
 5. Thetransformed host cell of claim 2 in which the glucuronide permease ΔNAis under the control of a second nucleotide sequence that regulates geneexpression in the host cell so that glucuronide permease is expressed inthe transformed host cell.
 6. The transformed host cell of claim 3 inwhich the glucuronide permease ΔNA is under the control of a secondnucleotide sequence that regulates gene expression in the host cell sothat glucuronide permease is expressed in the transformed host cell.